*Problem is increasing

*Problem is increasing
Salinity Impact on Crop Production Worldwide World Land Surface Area 150 x 10 6 km 2 Salt affected 9 x 10 6 km 2 (6%) Cultivated Land 15 x 10 6 km 2 * Salt affected 2 x 10 6 km 2 (13%) Irrigated Land 2.4 x 10 6 km 2 * Salt affected 1.2 x 10 6 km 2 (50%) *Problem is increasing Negative Impacts of Salinity on Agriculture Reduced yields on land that is presently cultivated Limited expansion into new areas Glycophytes vs halophytes - sweet plants and salt plants, respectively, by definition halophytes are “native flora to a saline environment” Quantitative difference - adaptation Nearly all salt tolerant plants are angiosperms, indicating polyphyletic origin, or halophytes are primitive genetic remnants of different families Salt tolerant species exist in 1/3 of the angiosperm families; however about ½ of the 500 halophytic species belong to 20 families, monocots - 45 genera in the Poaceae family and dicots - 44% of the halophytic genera are in the Chenopodiaceae (Atriplex, Salicornia and Suaeda) Most plants, including the majority of crop species, are glycophytes and cannot tolerate high salinity. Salt tolerance research is important basic plant biology,contributing to our understanding of subjects ranging from gene regulation and signal transduction to ion transport, osmoregulation and mineral nutrition.Additionally,some aspects of salt stress responses are intimately related to drought and cold stress responses. Plant salt tolerance studies thus contribute to understanding cross-tolerance

Salt tolerance research is important basic plant biology
Salt tolerance research contributes to our understanding of subjects ranging from gene regulation and signal transduction to ion transport, osmoregulation and mineral nutrition. Additionally,some aspects of salt stress responses are intimately related to drought and cold stress responses. Plant salt tolerance studies thus contribute to understanding cross-tolerance

Evolution of salt tolerance
Soil salinity almost always originates from previous exposure to seawater Although it is believed that for most of the Earth's history, the salt level of the oceans was much lower than now, all plant species that inhabit the seas, as well as a phylogenetically diverse groups of land plants, are capable of growth and reproduction at salinity levels near or above those found in the seas. This strongly supports the existence of a genetic basis for high-salinity tolerance within both sea and land plants. Plant Physiol. 135,

Sensitivity to salt occurs during all plant growth stages
germination NaCl inhibits both 1) germination and 2) growth למה?

Resistance to drought and salt stresses by neutrally charged osmolytes
Heat shock proteins LEA Compatible solutes protect the hydration shell

T6P inhibits growth when C-supply is limiting but is required for plants to utilize available carbohydrate. The authors demonstrate that Arabidopsis plants growing on trehalose-containing media are stunted as a result of accumulation of T6P, and the growth arrest can be rescued by feeding exogenous sucrose. The expression of many genes involved in stress responses was found to be correlated with the T6P level. Osmolytes/Osmoprotectants. Listed are common osmolytes involved in either osmotic adjustment or in the protection of structure. In all cases, protection has been shown to be associated with accumulation of these metabolites, either in naturally evolved systems or in transgenic plants

Salt stress

Secondary effects of NaCl stress
Reduced cell expansion and assimilate production – as during drought, adaptation includes reduction in cell expansion that affects photosynthate production Photosynthate production is reduced – carbon metabolism is salt sensitive Decreased cytosolic metabolism – metabolic poisoning, although enzymes of halophytes and glycophytes are equally sensitive to NaCl Production of ROS – products of photorespiration and mitochondrial respiration when electron flow is too great for the normal electron acceptors of metabolism, e.g. NADPH, resulting in the production of ROS A. spongiosa and S. australis are halophytes If the halophytes’ and glycophytes’ enzymes are equally sensitive to NaCl, why are the plants differentially sensitive to NaCl?

Na+ is cytotoxic, while K+ is an essential nutrient
Ion disequilibrium – Na+ rapidly enters the cell because the membrane potential inside is negative (~-120 to -200 mV), see slide Signal_transduction_of_Responses_to_Environment.ppt#17. Ionomics Na+ can accumulate to 102- to 103-fold greater concentration than in the apoplast, driven by the membrane potential, sea water 457 mM Na+ Na+ is cytotoxic, while K+ is an essential nutrient Ca2+ disequilibrium affects K+/Na+ selective uptake some plant species are also sensitive to Cl- Because Arabidopsis is a glycophyte and is very sensitive to salt, one might assume that this plant is not suitable for studying the mechanisms of salt tolerance. However, previous studies with cultured glycophytic plant cells indicated that these cells could be adapted to tolerate high concentrations of salt Cultured tobacco (glycophyte) cells are inhibited by 100 mM NaCl; however, after adaptation tobacco cells can grow in 500 mM NaCl Thus, the salt tolerance mechanism exists in glycophytes

Selective ion uptake and differential ion compartmentalization are main features that explain salt tolerance disparity between glycophytes and halophytes (Flowers et al., 1977 ; Greenway and Munns, 1980 ; Jeschke, 1984 ). Salinity affects nutrient acquisition by interfering with K+ uptake by carriers and channels. At the cellular level, intracellular ion sequestration into vacuoles for osmotic adjustment, strong ion selectivity in the cytosol (preference of K+ over Na+), and accumulation of compatible (non-toxic) organic solutes in the cytosol to equilibrate water potential across the tonoplast are widely accepted mechanisms contributing to salt tolerance (Greenway and Munns, 1980 ; Gorham et al., 1985 ). The sequestration of ions that are potentially damaging to cellular metabolism (e.g. Cl–, Na+) into the vacuole while maintaining high K+/Na+ ratios in the cytosol would provide the osmotic driving force required for water uptake in saline environments and, at the same time, provide plants with an efficient instrument for ion detoxification

Hamilton,E & Heckathorn, S (2001)
NaCl induces cytological hallmarks of programmed cell death in the wild-type yeast Nuclear fragmentation also IN PLANTS Bc2-2 protects Nuclear fragmentation (1 h) normal mitochondrion abnormal mitochondrion d) Nuclear fragmentation; e) vacuolation; f) coalescence of vacuolar and nuclear membranes; g) cell lysis. Hamilton,E & Heckathorn, S (2001) Plant Physiol. 126, IN PLANTS Mitochondrial adaptations to NaCl. Complex I is Protected by Anti-Oxidants and Small Heat Shock Proteins, whereas Complex II is Protected by Proline and Betaine.

NaCl Uptake into Roots and Movement in the Plant
Radial transport from the soil solution into roots is apoplastic/symplastic (epidermis and cortex), symplastic across the endodermis and then loaded into the xylem Na+ exclusion in shoot may be explained by K+/Na+ discrimination during xylem loading Radial transport may be regulated, i.e., Na+ and Cl- transport to the xylem is limited in epidermal and cortical cells, i.e., prior to the endodermis, but xylem loading is passive, plants can regulate K+/Na+ concentration in the xylem sap. Casparian strip ensures that all substances pass through at least one membrane before entering the stele Salt movement through the xylem is determined by the transpirational flux – moves through the xylem to the shoot Plants minimize exposure of meristematic cells to Na+ and Cl- - the lack of vasculature to the meristem reduces transport to these cells, mature leaves are ion sinks and may abscise Some halophytes deposit salt on the surface of leaves (sink) via glands or bladders

development of salt-tolerant crops (i.e. accumulation of salt)
Twenty years ago, Epstein argued for the development of salt-tolerant crops with truly halophytic responses to salinity in which the consumable part is botanically a fruit, such as grain or berries or pomes. In these plants, Na+ would accumulate mainly in their leaves and, because the water transport to the fruits and seeds is mainly symplastic, much of the salt would be screened from these organs. Thus, engineering the accumulation of salt in vacuolated cells, together with the active extrusion of Na+ from non-vacuolated cells (i.e. young and meristematic tissue),will allow the maintenance of a high cytosolic K+/ Na+ ratio. In combination with the enhanced production of compatible solutes…

Studying the Salt stress
1) Physiology of salt toxicity and salt tolerance. This includes cellular and metabolic responses to salt (Bohnert and Sheveleva, 1998 ; Hasagewa et al., 2000 ), as well as whole plant responses (Flowers et al., 1997; Greenway and Munns, 1980 ; Yeo, 1998 ). 2) Mechanisms of salt transport across cellular membranes and over long distances. This includes physiological and molecular characterization of ion transporters involved in salt uptake, extrusion, compartmentalization (Blumwald et al ; Schachtman and Liu, 1999 ). 3) Survey genes whose expression is regulated by salt stress (Zhu et al., 1997 ; Xiong and Zhu, 2001 ; Shinozaki and Yamaguchi-Shinozaki, 1997 ; Ingram and Bartels, 1996 ; Bray, 1997 ; Bohnert et al., 1995 ). This research is accelerated by using microarrays (Seki et al., 2001 ; Kawasaki et al., 2001 ; Bohnert et al., 2001 ). 4) Mutational analysis of salt tolerance determinants and salt stress signaling (Zhu, 2000 ; 2001a , b ; Xiong and Zhu, 2001 ).

Salt research approaches I
Comparative biochemistry (between species, treatments) osmolytes ROS ion compartmentation mechanisms (Na+ enters root cells mainly through various cation channels, particularly voltage-(in)dependent cation channels. Na+ and K+ Mutants (Up OR Down) Overexpression of individual components Complementation of yeast mutants inhibitors of salt adaptation in yeast

Functional Genomics of Plant Stress Tolerance
Complexity and Multigenicity of Stress Responses. 1. Variations on common physiological Themes. 2. Evolutionary Conservation of Stress Responses Mutants with altered sensitivity to osmotic/salt stress Mutants in stress signal transduction pathways using osmotically regulated promoter-reporter screening Identify Suppressors of Stress-responsive mutants

salt cress Unlike Arabidopsis leaf morphology, salt cress displays succulent-like leaves after salt exposure, measured as FW to dry-weight ratio. The development of a second layer of leaf palisade cells may contribute to this and also affect the rate of water loss from leaves. In addition, the stomatal density on salt cress leaves is twice that of Arabidopsis, although the stomatal index is nearly the same This may allow more efficient distribution of CO2 to photosynthetic mesophyll cells at low stomatal apertures. Plant Physiol. 135, 1718 The difference in salt sensitivity/tolerance may have resulted from differences in regulatory circuits or from salt tolerance genes. For example, the vacuolar Na+/H+ antiporter gene AtNHX1 is not as highly inducible in Arabidopsis as its homologous gene is in halophytes, and high level AtNHX1 expression driven by the strong CaMV 35S promoter could significantly improve Arabidopsis salt tolerance (Apse et al., 1999 ; Hamada et al., 2001 ; Shi and Zhu, 2002 ). At Book

The role of Potassium (K+)
Potassium affects the life of every living being. K+ role in plant growth is quite similar to that for humans. K+ is not an integral part of organic molecules in plants. K+ is important in many biochemical reactions, e.g. translocation of carbohydrates Under severe deficiency, plants will often develop visible symptoms: older leaf edges will turn brown, yield and quality decline. Sometimes, orange trees will drop their fruit; strawberries do not develop their sweet taste; corn stalks will break; tomatoes will be small and contain too much white tissue. Alfalfa will show typical yellowing along the outer margins of the leaves.

Salt stress impairs K nutrition
The membrane potential difference at the plasma membrane of plant cells is -140 mV, which favors passive transport of Na+ into cells, especially with high extracellular Na+ concentrations. Excess extracellular Na+ enters the cell through both the transporter HKT1 and non-selective cation channels/ transporters, which results in a decrease in the K+/Na+ ratio in the cytosol. Why? One way, possibly because of similarity between Na and K and the use of the same transporters, and the abundance of Na With increased concentration of NaCl in the medium, Na+ ↑ whereas K+ ↓. Analysis of ion content in seedlings transferred for 3d to high NaCl Adding calcium (Ca2+) to root growth medium enhances salt tolerance in glycophytes (6-8). Ca2+ sustains K+ transport and K+-Na+ selectivity in Na+-challenged plants (8) Science 280,

Dealing with Ion Toxicity
Because Na+ and K+have similar physic-chemical properties, high concentration of Na+ inhibits K+ uptake by the root. K+ uptake via Arabidopsis KUP1 is inhibited by >5 mM NaCl. Plants use both low and high affinity systems for K+ uptake (to match different soils). Sodium, once enters into the cytoplasm, inhibits many enzymes. This inhibition is also dependent on the K+ level in the cytoplasm Na+ are more damaging on the low affinity system that has low K+/Na+ selectivity. Under Na+ stress, it is necessary to use more selective high affinity K+ uptake system in order to maintain adequate . It is a general phenomenon that salt treatment of plants causes a decrease in cellular K+ content, which may be partly responsible for reduced growth and vigor under salt stress. KUP1 high-affinity potassium transport protein (Kim et al., 1998 ; Fu and Luan, 1998 ) (Fu and Luan, 1998 )

High-Affinity Potassium Transporter
AtKUP1 and AtKUP2 Complement Potassium Transport Deficiency in E. coli TK2463 Cells. Enhanced 86Rb+ Uptake in Transgenic Arabidopsis Suspension Cells Expressing AtKUP1

Na+ UPTAKE/EXTRUSION IN THE PLANT CELL
Plasma Membrane PPi H+ Na+ Na+ K+ H+ High-affinity K+ transporters V-PPase Na+ H+ Na+/H+ antiport Vacuole Na+ Na+ Tonoplast V-ATPase K+ Primary active P-ATPase uses the energy of ATP hydrolysis to pump H+ out of the cell generating an electrochemical H+ gradient. This proton motive force operates Na+/H+ antiporter which extrudes Na+ against its electrochemical gradient coupled with the movement of H+ into the cell along its electrochemical gradient. The primary active V-ATPase and V-PPase (pyrophosphatase) energize the tonoplast for secondary active transport of Na+ into the vacuole by the Na+/H+ antiporter. Plant cells lack Na+/K+ ATPase Ion ratios are altered by influx of Na+ thus plants maintain low cystolic Na+ [] and high cystolic K+/Na+ ratio Na+ [] is maintained in both halophytes and glycophytes at non-toxic levels by compartmentalization of Na+ into vacuoles which averts the deleterious affects of Na+ in the cytosol. Na+ can also enter via KUP/HAK/KT K+ transporters, cyclic-nucleotide-gated channels, glutamate-activated channels, LCT transporters and HKT transporters K+/Na+ selectiveVICs H+ ATP ATP K+/Na+ ratio H+ P-ATPase Adapted from Mansour et al. 2003

The plasma membrane proton H+ pump
Plants actively extract nutrients (NPK, etc) from the soil, and actively transport products of photosynthesis (such as sucrose) to parts of the plant that do not carry out photosynthesis (roots). The key enzyme in these processes is the plasma membrane H+-ATPase that pumps protons across the PM and thereby generates the proton and electrical gradient that is the driving force for secondary active transport executed by carriers and channels

Mechanisms of Salt Entry into Root Cells
Current evidence suggests that Na+ enters root cells through various cation channels that could be voltage-dependent or independent cation channels (VIC). Among them, VIC channels are considered the major route for Na+ entry (Amtmann and Sanders, 1999 Under normal conditions, the plasma membrane potential (MP) of root cells is -130 mV. A more negative potential would facilitate entry of the positively charged Na+ into cells. MP in plant cells is generated by ATPases, which pump H+ out of the cell creating electrochemical potential which facilitate the uptake of solutes. Some transporters affect salt sensitivity indirectly by altering MP as a result of regulation of ion flux. For example, in yeast trk mutants are defective in K+ uptake, the PM becomes hyperpolarized and this enhances the uptake of cations and rendered the mutants more sensitive to Na+, Li+, and low pH (Serrano et al., 1999 ). Membrane hyperpolarization enabled K+ uptake through other transporters. Interestingly, Ca2+ can reverse the salt sensitivity in the pmp3 mutant. PMP3 is a small hydrophilic protein predicted in the PM. It is not known how this protein can regulate membrane potential. PMP3 is homologous to the Arabidopsis proteins RCI2A and RCI2B (Medina et al., 2001 ; Nylander et al., 2001 )

Sodium Fluxes through Nonselective Cation Channels in the Plasma Membrane of protoplasts from Arabidopsis Roots To distinguish Na+ influx catalyzed by NSCCs from that catalyzed by K+-selective and Ca2+-selective channels, experiments with the K+ and Ca2+ channel blockers tetraethylammonium (TEA+) and verapamil were carried out (Fig. 2). The addition of 10 mM TEA+ to a background of 50 mM NaCl did not decrease currents; in fact, it usually slightly increased the inward current, probably because TEA+ permeates the NSCCs (see below). In the same conditions, 100 µM verapamil slightly decreased the inward currents (by up to 30% of the current amplitude) at voltages How do you distinguish Na+ influx catalyzed by NSCCs from that catalyzed by K+-selective and Ca2+-selective channels? Instantaneous currents through the plasma membrane of Arabidopsis root protoplasts in response to voltage-clamp steps from 160 to 80 mV (holding potential = 70 mV). Solutions contained 10, 20, or 100 mM NaCl. To distinguish Na+ influx catalyzed by NSCCs from that catalyzed by K+-selective and Ca2+-selective channels, experiments with K+ and Ca2+ channel blockers Plant Physiol. 2002 February; 128(2): 379–387.

Ion Homeostasis Transport Determinants
Plasma membrane: Influx - Na+ influx is passive (nonselective cation channel(s) (NSCC), HKT1 transport system, leak through K+ uptake systems; Cl- uptake is active (because of the inside negative potential across the plasma membrane) Efflux – Na+ efflux is active, H+ driven Na+ antiporter SOS1, proton gradient is established by the plasma membrane (P-type) H+-ATPase, note the ∆pH Tonoplast: transport into the vacuole Na+ - influx, H+ driven Na+ antiporter NHX family, proton gradient is established by the tonoplast (V-type) H+-ATPase and pyrophosphatase, ∆pH Ion Homeostasis Transport Determinants The membrane potential difference at the PM of plant cells is -140 mV, which favors passive transport o f Na+ in to cells,especially with high extracellular Na+ concentrations. Excess extracellular Na+ enters through both the transporter HKT1 and non-selective cation channels/transporters, which results in a decrease in the K+/Na+ ratio in the cytosol. The high affinity K+ transporter HKT1 appears to act as a low affinity Na+ transporter

Osmotic Adjustment and Ion Comparmentalization
Cells expend ~50% of their total energy to maintain gradients of ions across membranes. The electrochemical potential of these ion gradients represents stored energy. Plants and fungi are similar in that they use proton (H+) gradients as the "currency" with which to mediate transport of ions K+(Na+) H+ K+(Na+) K+ Na+/H+ K+ polyols proline betaine trehalose cp pH 7.5 Na+ *-scavenging pH 5.5 mt Tonoplast perox NaCl↑ Plasma Membrane -120 to mV ATP ATP H+ Na+ Cl- Ca2+ pH 5.5 H+ PPi +20 to +50 mV H+ Na+ H+ Ca2+ Ca2+ ATP Na+ H+ H+ H2O H+ Cl- Cl- ATP When plant and yeast cells are dehydrated, the water reservoir in the vacuole can compensate the water deficit in the cytosol, but its volume is consequently reduced while its surface area is unchanged. Fragmenting the vacuole easily solves the problem, and the more vesicles formed, the smaller the volume, while maintaining the membrane area. Na+ exclusion in shoots of some plants may be explained by K+/Na+ discrimination during xylem loading The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. Algae produce DMSP to protect themselves from the negative effects of high salinity and freezing. DMSP is also formed in some higher plants that are tolerant to drought, frost and salt stress Cl- Ca2+ H+ Ca2+ Ca2+ Cl- Ca2+ Inositol H2O Na+ Cell volume increases 10- to 100-fold during growth and development due almost entirely to an increase in the vacuole size, i.e., water uptake into the vacuole drives cell expansion Na+ and Cl- compartmentalization in the vacuole is a necessary component of osmotic adjustment, net uptake of these ions across the plasma membrane is restricted and organic osmolytes mediate osmotic adjustment in the cytosol

Model of H+ pumps and transporters found in the plant vacuolar membrane
A) H+-ATPase (1) and H+-PPase (2) transport protons (H+) into the vacuolar lumen. Organic and inorganic anions (A) enter the vacuole via channels (4) to electroneutralize, allowing the generation of a pH gradient. This (proton electrochemical gradient [PEG] drives secondary active accumulation of organic and inorganic cations into the vacuole via H+/cation antiporters (3), with osmotically accompanying water. B, Ectopic expression of cation/H+ antiporters (3) in the vacuolar membrane sequester higher amounts of cations through the utilization of the existing PEG generated via the two H+ pumps (1 and 2). C, Reduced H+-pumping activity in the det3 mutant. H+-ATPase activity (1) in the det3 mutants is diminished. A reduction in the PEG activities (3 and 4) across the vacuolar membrane. D, Ectopic expression of AVP1. Transgenic plants with enhanced AVP1 (2) have an enhanced PEG. This altered PEG increases transport activities (3 and 4) across the vacuolar membrane.

Transgenic tomato with vacuolar Na+/H+ antiport (AtNHX1) allowed them to grow in 200 mM NaCl
vNa/H wt

Ca2+ in Na+ stress An important determinant for salt tolerance relevant to Na+ and K+ homeostasis is Ca2+. Increased Ca2+ supply has a protective effect on plants under Na+ stress. Early experiments did not distinguish whether Ca2+ acted extracellularlly or intracellularly. Recently, altered cellular Ca2+ homeostasis showed that internal/cytosolic Ca2+ is important to salt sensitivity regulation. 1) e.g., expression of AtACA4 that codes for a vac- Ca2+-ATPase in yeast increased their salt tolerance 2) Arabidopsis vacuolar Ca2+/H+ antiporter gene CAX1, when overexpressed, increased sensitivity to ionic stress. These transgenic plants appeared Ca2+-deficient despite a higher total Ca2+ content (Hirschi,1999) hkt1 mutation suppressed Na+ sensitivity of sos3 mutants, but not in low Ca2+ (0.15 mM), suggesting an alternative Na+-influx system, different from AtHKT1, that is hampered by high Ca2+ (2 mM) but is the prevalent Na+ entry pathway at low external Ca2+.

CAX1 Expression Disturbs Normal Vigor
this study shows perturbed growth by constitutive expression of a single transport protein. The CAX1-transgenics displayed altered phenotypes and increased stress sensitivity Plant Cell, Vol. 11, CAX1-expressing lines CAX1-expressing lines after several weeks in the greenhouse. & (D) Size of CAX1-expressing plants. in the background is expressing CAX1 in the antisense confirmation. This plant is the same size as control plants (E) The sense roots are significantly stunted. (F) Leaf of 10-week-old vector control plant grown for 2 weeks without Ca2+. (G) Leaf of 10-week-old CAX1-expressing plant given Ca2+ supplementation.

Plant phenotypes of altered expression of H+ pumps and H+/cation antiporters
Expression of CAX1 in tobacco causes apical burning and other growth defects associated with calcium deficiencies. B, CAX2 makes plants more tolerant of Mn. The CAX2 sense- and antisense- plants grown in MnCl2. Control (C) and AtNHX1 transgenic tomato D) growing in the presence of 200 mM NaCl. E, det3 and control Arabidopsis plants grown in soil. F, Control and transgenic AVP1 lines after recovery from 10 d of drought stress. Gaxiola, R. A., et al. Plant Physiol. 2002;19:

Ion Sensitivity of CAX1-Expressing Plants
Two vector control plants are shown at left and two CAX1-expressing plants (35S::CAX1) at right. (A) Plants grown in standard media immediately after transfer to various media (pretreatment). (B) Plants transferred to standard media and grown for 10 days. (C) Plants transferred to standard media supplemented with 50 mM MgCl2 and grown for 10 days. (D) Plants transferred to standard media supplemented with 100 mM KCl and grown for 10 days. (E) Plants transferred to standard media supplemented with 50 mM NaCl and grown for 10 days. (F) Plants transferred to standard media supplemented with 100 mM CaCl2 and grown for 10 days. (G) Plants transferred to standard media without Ca2+ and grown for 10 days. (H) Plants transferred to standard media supplemented with 50 mM MgCl2 and 2 mM CaCl2 and grown for 10 days. (I) Plants transferred to standard media supplemented with 100 mM KCl and 2 mM CaCl2 and grown for 10 days

Salt stress in yeast: the HOG pathway (High Osmolarity Glycerol) of S
Salt stress in yeast: the HOG pathway (High Osmolarity Glycerol) of S.cerevisiae Mol. Cell. Biol. 17, The yeast HOG1 signal transduction pathway contains two independent osmosensors. The first is a two-component signal transducer, whereas the second osmosensor, Sho1p, is a transmembrane protein with a cytoplasmic SH3 domain. Under normal osmotic conditions the transmembrane his-kinase Sln1p transfers a phosphate to Ssk1. Phosphorylation of Ssk1p inhibits Ssk1p-mediated activation of Ssk2p and Ssk22p MAPKKKs. Increased osmolarity inactivates Sln1p his-kinase and unphosphorylates Ssk1p activating the Ssk2p and Ssk22p MAPKKKs, which in turn activate Pbs2p. High osmolarity causes Sho1p interaction with and activation of Pbs2p. The activated Pbs2p phosphorylates and activates Hog1p Activation of Hog1p leads to induction of genes for adaptation to high-osmolarity stress, including GPD1, CTT1 and HSP12 primary sensors of osmotic stress, the Sln1p-Ssk1p two -component proteins, are involved in sensing oxidative stress specifically induced by hydrogen peroxide and diamide, but not by other oxidants [The best-characterized two-component histidine kinase is the Saccharomyces cere6isiae osmosensor SLN1.Together with the YPD1-SSK1 response regulator,this ‘two-component’ signal unit regulates the high-osmolarity glycerol (HOG)MAPK cascade,resulting in the production of glycerol to survive osmotic stress.In Arabidopsis,a histidine kinase gene,AtHK 1,was isolated by PCR using degenerate primers.This kinase is structurally related to SLN1. REV in Physiol.Plant.112,2001 J-K Zhu] The yeast HOG1 signal transduction pathway contains two independent osmosensors. The first is a two-component signal transducer and is actually composed of three proteins (Sln1p, Ypd1p and Ssk1p), whereas the second osmosensor, Sho1p, is a transmembrane protein with a cytoplasmic SH3 domain. Under normal osmotic conditions, the transmembrane histidine kinase Sln1p is catalytically active and transfers a phosphate by a phosphorelay mechanism, via Ypd1p, to the response regulator protein Ssk1p. Phosphorylation of Ssk1p appears to inhibit the ability of Ssk1p to activate the Ssk2p and Ssk22p MAPKKKs. In the presence of increased osmolarity, the Sln1p histidine kinase is inactivated and unphosphorylated Ssk1p activates the Ssk2p and Ssk22p MAPKKKs, which in turn activate Pbs2p. Alternatively, in the presence of a high osmolarity stimulus, Sho1p interacts with and activates Pbs2p. The activated Pbs2p then phosphorylates and activates Hog1p. Activation of Hog1p leads to the induction of transcription of genes required for adaptation to high-osmolarity stress, including GPD1, CTT1 and HSP12 the HOG1 pathways for adaptation to hyperosmotic stress and the calneurin pathway for ionic stress. In yeast, Na +, K+ and Ca2+ and the pheromone response are regulated by calcineurine; mutants at the calcineurin locus are sensitive to Na+ and Li+.

Membrane stretching in salt stress
In yeast, hyperosmolarity can be sensed by a two-component system composed of the SLN1 His kinase, the YPD1 phosphorelay intermediate, and the SSK1 response regulator, leading to the activation of the HOG1 MAPK pathway. The Arabidopsis (Arabidopsis thaliana) SLN1 homolog, AtHK1, is able to suppress the salt-sensitive phenotype of the yeast double-mutant sln1 sho1 , which lacks both yeast osmosensors (Urao et al., 1999 ). However, direct evidence for a role of AtHK1 as an osmosensor in plants is still lacking. Although it could interact with the phosphorelay AtHP1 in the yeast two-hybrid system, no interaction was observed between AtHP1 and the response regulators (Urao et al., 2000 ). Another His kinase, CRE1, which was identified as a cytokinin receptor, is also able to complement the yeast sln1 mutant in the presence of cytokinin (Inoue et al., 2001 ). Interestingly, a recent work reported that SLN1 and CRE1 perceive the osmotic signal by turgor sensing in yeast (Reiser et al., 2003 ). It was shown that the integrity of the periplasmic region of SLN1 is essential for its sensor function. This suggests that osmotic stress may trigger a conformational change of SLN1 due to a stress-induced modification of the cell wall-plasma membrane interaction. It is tempting to speculate that a similar turgor-sensing mechanism might regulate hyperosmotic signaling in plants. On the other hand, the involvement of receptor-like kinases (RLK) in osmosensing has been suggested by the increased osmotic stress tolerance induced by overexpression of the tobacco (Nicotiana tabacum) NtC7 (Tamura et al., 2003 ). At least two of the Arabidopsis histidine kinase genes, ARABIDOPSIS THALIANA HISTIDINE KINASE1 (ATHK1) and CYTOKININ RESPONSE1 (CRE1), complement sln1 deletion mutants of yeast [15,17] and CRE1 can also respondto changes in turgor pressure when expressed in yeast [15]. Yet, these proteins have not been shown to function asosmosensors in plants

Two-component signaling system

Ion homeostasis after salt (NaCl) adaptation.
HOG1 pathways for osmotic homeostasis for (i) low osmolarity sensor SHO1 or (ii) high osmo sensor. SLN1: SLN1 SSK1 PBS2HOG1 or SHO1 PBS2HOG1. Stress adaptation effectors are those that mediate ion homeostasis, osmolyte biosynthesis, toxic radical scavenging, water transport. Both pathways converge at PBS2 leading to transcriptional activation of glycerol biosynthetic genes Indicated are the osmolytes and ions compartmentalized in the cytoplasm and vacuole, transport proteins responsible for Na and Cl- High NaCl causes cytosolic accumulation of Ca2+ and this signals stress responses that are either adaptive or pathological. Determinants of plant stress tolerance have been identified, by functional complementation of osmotic yeast mutants. A MAPK has been identified from Pisum with 47% sequence identity to Hog1p, which is the MAPK in the yeast osmoregulatory pathway that controls glycerol accumulation. ATHK1 resembles the yeast osmosensor SLN1 that functions both as the sensor and receiver of the phospho-relay system that initiates the two-component HOG MAPK pathway that mediates hyperosmotic stress tolerance. The calc`ineurin pathway in yeast is essential for regulation of key life cycle processes other than salinity tolerance, suggesting that salt stress response and adaptation are integrated into cellular homeostasis PsMAPK functionally suppressed salt-induced cell growth inhibition of hog1. Combinations of Arabidopsis proteins ATMEKK1 (MAPKKK) and MEK1 (MAPKK), or ATMEKK1 and ATMKK2 (MAPKK) suppressed growth defects of pbs2 (wild-type allele encodes the MAPKK of the HOG pathway), implicating these as functional components of an osmotic stress MAP kinase cascade High NaCl causes cytosolic accumulation of Ca2+ and this signals stress responses that are either adaptive or pathological. NaCl  Ca2+  CDPKs/MAPKs

Activation of two MAPK cascades in yeast
HOG MAP kinase cascade Science 299:1025-7 (Left) The mating cascade is activated when the cell's a-factor receptor receives the a-factor pheromone from an expectant partner. The receptor is associated with a G protein, and interaction with pheromone frees the G protein. that exposes a surface which binds to the scaffold Ste5. (Right) High osmolarity cascade is activated by the membrane protein Sho1. Under high-salt conditions, Sho1 exposes a surface that binds to the scaffold Pbs2. (Center) Ste20 is an active kinase tethered to the membrane. Prot-G recruits Ste5 to the membrane, where Ste20 triggers the mating cascade. Sho1 recruits Pbs2 to the membrane, where Ste20 triggers the osmolarity cascade. The Pbs2 scaffold has two bound kinases and akinase domain. Fus3 and Hog1 are called MAPKs, Ste7 and Pbs2 are MAPKKs, and Ste11 is a MAPKKK. When yeast cells are exposed to conditions of high extracellular osmolarity, the HOG MAP kinase cascade is activated, producing a variety of cellular responses, including glycerol production. Under conditions of normal extracellular osmolarity, the kinase cascade is inactivated. Yeast cells also contain a second transmembrane osmosensor, Sho1p, which activates the Pbs2p kinase directly in response to high osmolarity The HOG pathway plays a critical role in the yeast osmostress response and is composed of a signal transducer (Sln1p, Ypd1p, and Ssk1p) and an MAP kinase cascade (Ssk2p/Ssk22p, Pbs2p, and Hog1p) (Figure 5). Under conditions of normal extracellular osmolarity, the Sln1p histidine kinase is autophosphorylated, and a relay system sequentially transfers the phosphate from Sln1p to Ypd1p and finally to Ssk1p (Posas et al., 1996 ). Phosphorylated Ssk1p cannot activate the Ssk22p and Ssk2p MAPKKKs and, as a result, signaling via the Hog1p kinase is inhibited. When extracellular osmolarity is high, the Sln1p kinase is inhibited, the active, unphosphorylated form of Ssk1p interacts with the Ssk22p and Ssk2p MAPKKKs, and the HOG MAP kinase cascade is turned on. Activation of the Hog1p kinase at the end of this cascade results in a variety of cellular responses, including production of intracellular glycerol. Pbs2p (MAPKK) and Hog1p (MAPK) in the pathway are thought to be dephosphorylated and down-regulated by the serine/threonine phosphatases Ptc1p and Ptc3p (Maeda et al., 1994 ) and the tyrosine phosphatases Ptp2p and Ptp3p

SH3 domains (AtSH3Ps Partially Complement a Salt-Sensitive, Endocytosis-Deficient Yeast Mutant)
The basic fold of SH3 domains contains five anti-parallel beta-strands packed to form two perpendicular beta-sheets. The ligand-binding site consists of a hydrophobic patch that contains a cluster of conserved aromatic residues and is surrounded by two charged and variable loops Domain Binding and Function Src-homology 3 (SH3) domains generally bind to Pro-rich peptides that form a left-handed polyPro type II helix, with the minimal consensus Pro-X-X-Pro. Each Pro is usually preceded by an aliphatic residue. Each of these aliphatic-Pro pairs binds to a hydrophobic pocket on the SH3 domain. Class I and 2 of SH3 domains have been defined which recognize RKXXPXXP and PXXPXR motifs respecitvely

Activation of protein kinases by hyperosmotic stress

Osmotic stress-activated protein kinases in plants
MAPKs are induced by osmotic stress (salt and drought) stress These data mean that each stress level produces its own unique combination of signals (signal signature) that activates the appropriate graded response. The fact that different salt ranges activate different pathways supports the concept that stress is detected by different receptors responding over those limited ranges, in a manner similar to the osmo-sensors in yeast FEBS 498;172 (2001) Two MAPKs are activated in an in-gel assay. One is activated at moderate concentrations, responding in a dose-dependent way, peaking at 500 mM NaCl, whereas the other was only activated at very high concentration, starting at 500 mM NaCl The fact that different salt ranges activate different pathways supports the concept that stress is detected by different receptors responding over those limited ranges, in a manner similar to the osmo-sensors in yeast

Salt signaling in yeast & plants
In addition to MAPK pathway, yeast has another pathway specific for high NaCl, which includes calcineurin, a phosphatase dependent on Ca2+, and calmodulin. Therefore, it is possible that external high NaCl increases intracellular Ca2+, which then causes calmodulin to transmit signals to other, downstream components, such as calcineurin The SLN1 branch of the HOG pathway is stimulated by turgor reduction An Arabidopsis GSK3/shaggy-Like Gene that Complements Yeast Salt Stress-Sensitive Mutants Is Induced by NaCl and Abscisic Acid Plant Physiol. 119 :1527 encode kinases Sln1 and Sho1 have distinct cellular distributions. (A) Architecture of the SLN1 and SHO1 branches of the HOG pathway. (B) Either the SLN1 or SHO1 branch is sufficient to survive on high osmolarity. GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. Osmosensor molecules, such as Sln1p and Sho1p, which are located on the plasma membrane, initiate the signaling pathway (Ota and Varshavsky, 1993 ). The signal then reaches various kinases such as Ssk1p, Pbs2p, and Hog1p (Maeda et al., 1994 , 1995 ; Posas et al., 1996 ). In addition to the MAP kinase pathway, yeast has another signal transduction pathway that is specific for high NaCl stress. This pathway includes calcineurin, a phosphatase dependent on Ca2+, and calmodulin (Nakamura et al., 1993 ; Mendoza et al., 1994 ; Wieland et al., 1995 ). Therefore, it is possible that external high NaCl stress increases intracellular Ca2+ concentration, which then causes calmodulin to transmit signals to other, downstream components, such as calcineurin SLN1 and SHO1 branches in the HOG pathway respond independently to osmotic status of the environment and are apparently redundant. However,in the SLN1 branch, a transmembrane (TM) histidine kinase Sln1 serves as an osmosensor, and transmits the signal through the Sln1–Ypd1–Ssk1 multistep phosphorelay to the redundant pair of kinases Ssk2 and Ssk22. In contrast, another TM protein (Sho1) serves as a facilitator of signaling module assembly that includes Pbs2, Ste11, Ste20, and Cdc42

Activation of distinct lipid and MAPK signalling pathways by osmotic stress
Activation of different receptors, dependent on the stress level when 100 mM NaCl is then stressed by additional salt, the same signaling pathways were still activated in the same response pattern. This is unlikely, if they detect salt concentrations  thus, they detect a consequence of increased salt, such as loss of turgor. Thus, osmo-sensors are stretch receptors that respond to changes in membrane pressure When Chlamydomonas was grown in 100 mM NaCl and then stressed by additional salt, the same signalling pathways were still activated in the same response pattern [89]. This is interesting because the osmo-sensors and their signalling pathways are now responding to much higher salt concentrations. Since this seems unlikely, if they detect salt concentrations, we can conclude that they detect a consequence of increased salt, such as loss of turgor. This suggests that the osmo-sensors are stretch receptors that respond to changes in membrane pressure and so remain operative irrespective of whether the cells are osmo-adapted or not. However, the change in turgor when cells are shifted from 100 to 200 mM salt should be less than when shifted from 1 to 100 mM salt. Although signalling in adapted cells remained the same, all optima were shifted to higher concentrations and in general less signal was formed. Proprioreceptors – These are receptors which respond to stretch or pressure. Examples of these receptors are found within the walls of our gastrointestinal tract and blood vessels. On the one hand, proprioreceptors within our gastrointestinal tract sense food particles and signal the initiation of peristalsis (i.e., rhythmic contraction of the G/I tract to propel food and sustain the digestive process). On the other hand, proprioreceptors within some of our blood vessels sense changes in blood pressure which are subsequently reported to the vital reflex centers of the CNS. As we’ll see later, the perception of blood pressure change is one of the body’s highest physiological priorities. More on MAPK MAPKK in salt stress in MAPKs-Plants Sig slide 13 CDPK, calmodulin-like domain protein kinase; DAG, diacylglycerol; DGPP, diacylglycerol pyrophosphate; IP3, inositol 1,4,5-trisphosphate; L-PA, lyso-PA; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PI3K, phosphoinositide 3-kinase; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D.

Algorithm for discovering stress tolerance determinants

Comparison of Salt Sensitivity in sos mutants on Vertical Plates by Using the Root-Bending Assay
How do we know that sos1-sos3 are different allelles? 1) Complementation 2) different stress conditions (other ions) [NEXT SLIDE] Five-day-old seedlings were transferred from normal MS medium to high NaCl, and the seedlings (with roots upside down) were grown for 7 d. Continued growth on salt plates results in bending of roots due to gravitropism; thus,lack of root bending is a visual sign of inhibition by NaCI Genetic analysis indicates that SOS1 is epistatic to SOS2 and SOS Zhu Plant Phys 2000

Sensitivity of salt sensitive mutants to other salts
sos3 but not sos1 is fixed by high Ca WT, sos1, and sos3 mutants were exposed to high-salt (100 mM NaCl) or low-K+ (20 µM K+) stress

Complementation of sos3 by the wild-type SOS3 : SOS3 Binds 45Ca2+.
SOS3 encodes an EF hand–type Ca2+ binding protein with an N-myristoylation domain Similar to B-subunit of calcineurin (type 2B) protein phosphatase G2A (N-myristoylation ) Mutation Abolishes SOS3 Function in Plant Salt Tolerance but not Ca2+ binding SOS3 is a calcium binding protein with an N-myristoylation signature sequence Plant Cell, Vol. 12,

sos1 Plants Cannot Grow with Low K+
suggests the mutant may be deficient in high-affinity K + uptake sos1 needs high levels of K+ to grow suggesting that the mutant may be deficient in high-affinity K + uptake The K +content in the wild type did not decrease to <3%of the dry weight,while in sos1 it de-creased to ~1%, indicating that K +deficiency occurs in NaCI-treated sos1 plants. The observation that sos1 needs high levels of K +to grow indicates that the mutant may be deficient in high-affinity K + uptake. K+ content in NaCI-treated sos1 seedlings was measured to determine whether the mutant was deficient in K +.After 24 hr of exposure to various levels of NaCI,the K +content was decreased in both sos1 and the wild-type plants. More importantly,this decrease was greater in sos7 at all NaCI concentrations (Figure 11).The K +content in the wild type did not decrease to <3%of the dry weight,whereas in sos1 it de-creased to ~1%.The results demonstrate that K +deficiency occurs in NaCI-treated sos1 plants. (A)Plants on 20 mM K +. (B)Plants on 200 nM K*.

Callus Tissue Derived from sos1 Is Hypersensitive to NaCI.
Sodium extrusion is done by Na+/H+ antiporters in PM. The PM-localized Na+/H+ antiporter is SOS1. Mutations in SOS1 rendered the mutant plants very sensitive to Na+. Overexpressors had a lower Na+ content in the shoot upon treatment with Na+

Sensitivity of sos2 and sos3 seedlings to Salts
wt NaCl CsCl NaCl KCl KCl Mannitol sos2 LiCl CsCl LiCl Four-day-old seedlings were transferred to MS medium or MS media supplemented with various concentrations of NaCl, KCl, LiCl, or CsCl. Root elongation after 7 days is presented as a percentage relative to elongation on MS medium. Filled circles, wild type; open circles, sos2.

Mannitol stress sos1 Mutants but Not sos2 Mutants Are Hypersensitive to Mannitol Stress.

Salt stress is perceived by an unknown receptor (
Salt stress is perceived by an unknown receptor (?) at the plasma membrane (PM). It induces Ca2+, which is sensed by SOS3 that changes its conformation in a Ca2+-dependent manner and interacts with SOS2. This interaction relieves SOS2 of its auto-inhibition and results in activation of the enzyme. Activated SOS2, in complex with SOS3 phosphorylates SOS1, a Na+/H+ antiporter resulting in efflux of excess Na+ ions. SOS3–SOS2 complex interacts with and influences other salt mediated pathways resulting in ionic homeostasis. This complex inhibits HKT1 activity (a low affinity Na+ transporter) thus restricting Na+ entry into the cytosol. SOS2 also interacts and activates NHX (vacuolar Na+/H+ exchanger) resulting in sequestration of excess Na+ ions, further contributing to Na+ ion homeostasis. CAX1 (H+/Ca+ antiporter) has been identified as an additional target for SOS2 activity reinstating cytosolic Ca2+ homeostasis

Signaling pathways that regulate expression and activity of ion transporters to maintain low cytoplasmic Na+. The Na+ and hyperosmolarity are each perceived by unknown sensors

vacuolar antiporter AtNHX1
Induction of AtNHX1 expression by salt is not affected in sos1, sos2 or sos3 mutants. However, mutations that cause ABA deficiency or the ABA-insensitive1 (abi1) (but not the abi2) partially disrupt AtNHX1 induction by salt stress This suggests that an SOS-independent, ABA-dependent pathway regulates the expression of the vacuolar antiporter in response to salt stress (slide 53). However, the SOS pathway regulates the activity of vacuolar Na+/H+ antiporters

Functional demarcation of salt and drought stress signaling pathways.
Plants rarely experience stress from a single environmental source

Salt stress studies/research categories
1)Physiology of salt toxicity and salt tolerance. Cellular and metabolic responses to salt inc. whole plant responses. 2) Salt transport mechanisms across membranes & over long distance Physiological and molecular characterization of various ion transporters involved in salt uptake, extrusion, compartmentalization, and in the control of long distance transport. 3)Survey of genes whose expression is regulated by salt stress. This is being accelerated by using gene chips and cDNA microarrays. 4)Mutational analysis of salt tolerance determinants and salt stress signaling Classical and suppressor mutagenesis

hkt1-1 and hkt1-2 mutations suppress the NaCl hypersensitive phenotypes of sos3-1
suppressor mutations of the salt-sensitive Arabidopsis sos3 mutant, which hyperaccumulates Na+ seedlings were transferred to fresh medium. Root growth +/- NaCl after 6 days shoot growth and anthocyanin accumulation on medium with 75 mM NaCl after 15 days sos3 mutant hyperaccumulates Na+ T-DNA lines derived from sos3 mutant identified HKT1 transporter as suppressor of both the Na+ sensitivity and Na+ hyperaccumulation of the sos3 mutant, demonstrating that HKT1 is an entry system for Na+.

AtHKT1 is a salt tolerance determinant that controls Na+ entry into plant roots (PNAS 98 (24): NOV ) Extragenic Arabidopsis mutations that suppress NaCl hypersensitivity of sos3-1 were identified The sos3- hkt1-1 mutation can suppress the Na+ sensitivity of sos3-1 and reduce the intracellular accumulation of Na+. Moreover, sos3-1 hkt1-1 were able to maintain [K+](int) in hign NaCl and exhibited higher intracellular ratio of K+/Na+ than the sos3-1 mutant. hkt1 suppressed the Na+ hypersensitivity of sos3-1 much less when grown in low Ca2+ and abrogated the growth inhibition of the sos3-1 caused by K+ deficiency on low Ca2+ Thus, AtHKT1 is a salt tolerance determinant that controls Na+ entry and high affinity K+ uptake. The hkt1 mutation revealed the existence of another Na+ influx system(s) whose activity is reduced by high [Ca2+](ext). Na+ uptake across the plasma membrane is attributed to low Na+ permeability of transporters of the essential K+ nutrient. Na+ uptake across the plasma membrane has been attributed to low Na+ permeability properties of systems that transport the essential nutrient K+ (3, 10, 11). Transport systems that have high affinity for K+ but also have low affinity for Na+ include inward rectifying K+ channels (KIRCs) like AKT1, outward rectifying K+ channels (KORCs), and the KUP/HAK family of K+–H+ symporters (3, 11, 12). However, there have been some suggestions that Na+ influx may be mediated also by low affinity cation or nonspecific cation transport systems (1, 3, 10, 11). The high affinity K+ transporter (HKT1), low affinity cation transporter (LCT1), and nonselective cation channels are considered to be the most likely specific transport systems that mediate high Na+ influx (10, 11, 13–16).

Drought-and salt-tolerant plants by overexpression of the AVP1 H1-pump
WT AVP1-1 AVP1-2 after rewatering Cells expend as much as 50% of their total intracellular energy reserves to maintain gradients of ions across their membranes. The electrochemical potential of these ion gradients represents stored energy. Plants and fungi are similar in that they use proton (H+) gradients as the "currency" with which to mediate transport of ions. The simplicity of the vacuolar H+-PPase structure makes it an excellent candidate for manipulating the proton gradients in plants. Transgenic plants overexpressing AVP1 (AVP1OX) a gene that encodes the vacuolar H+-pyrophosphatase in Arabidopsis, are more salt- and drought-tolerant than their control counterparts SOS1 overexpression confers salt tolerance (Nature Biotech 21:81-85). In order to establish if there is an epistatic effect of AVP1 overexpression over the sos1 mutation, we crossed sos1-1 mutants with two AVP1OX lines. The driving hypothesis postulated that the enhanced vacuolar Na+ sequestration capacity displayed by AVP1OX plants could rescue the sos1 salt sensitivity. All the AVP1OX/sos1-1 lines tested were as sensitive to 50 mM NaCl as the original sos1 mutants. These results are consistent with an epistatic effect of the sos1 mutation over AVP1 overexpression in regard to salt tolerance ( Arabidopsis thaliana plants engineered to overexpress the vacuolar H+-pyrophosphatase AVP1 have enhanced tolerance to salinity and drought stress. The enhanced tolerance is most easily explained by an enhanced uptake of ions into their vacuoles. Presumably, the greater AVP1 activity in vacuolar membranes provides increased H+ to drive the secondary active uptake of cations into the vacuole. A compensatory transport of anions is expected in order to maintain electroneutrality. The resulting elevated vacuolar solute content would allow for greater osmotic adjustment capacity permitting plants to survive under conditions of low soil water potentials. salt drought AVP1 transgenic plants show that increasing the vacuolar proton gradient results in increased solute accumulation and water retention. Presumably, the greater AVP1 activity in vacuolar membranes provides increased H+ to drive the secondary active uptake of cations into the vacuole sequestration of cations in the vacuole reduces their toxic effects.

Ion Homeostasis Transport Determinants
Plasma membrane: Influx - Na+ influx is passive (nonselective cation channel HKT1 transport system, leak through K+ uptake systems; Cl- uptake is active (because of the inside negative potential across the plasma membrane) Efflux – Na+ efflux is active, H+ driven Na+ antiporter SOS1, proton gradient is established by the plasma membrane (P-type) H+-ATPase, note ∆pH Tonoplast: Na+ - influx, H+ driven Na+ antiporter NHX family, proton gradient is established by the tonoplast (V-type) H+-ATPase and pyrophosphatase, ∆pH

Salt Stress Signaling that Regulates Na+ Ion Homeostasis
Model predicts: Positive regulation: SOS1 AtNHX1, 2 and 5 (post-transcriptional?) [Na+]ext↑ → [Ca2+]cyt↑ → SOS3 → SOS2 Negative regulation: AtHKT1 [Ca2+]ext blocks Na+ uptake through NSCC vacuolar Na+/H+ antiporter gene Na+ transporter that controls Na+ entry into roots Non-selective calcium channel The activated SOS pathway and outputs of the pathway are targets for bioengineering of salt tolerance Ca2+ channel – two pore channel (α subunit of L-type), activated by hyperosmotic stress SOS3 – Ca2+-binding protein SOS2 – serine/threonine kinase that is activated by interaction with Ca2+-SOS3 SOS3-SOS2 complex phosphorylates SOS1 to activate its Na+/H+ antiporter activity. SOS3-SOS2 complex induces the expression of SOS1 through some yet unknown transcription factor. Does the SOS pathway regulate AtNHX family antiporters at the post-transcriptional level?

Generic pathway of salt, drought and cold stress
Salt and drought disrupt the ionic and osmotic equilibrium of the cell resulting in stress. This triggers the process, to reinstate ionic and osmotic homeostasis leading to stress tolerance. Stress imposes injury on cellular physiology and result in metabolic dysfunction. This injury imposes a negative influence on cell division and growth. This is an indirect advantage to the plant as reduction of leaf expansion reduces the surface area of leaves exposed for transpiration and thereby reducing water loss Stress injury and ROS from stress also trigger detoxification signaling by activating genes for damage control and repair, leading to stress tolerance. Cold stress mainly exerts its malicious effect by disruption of membrane integrity and solute leakage.

Plant Drought and Salt Stress Tolerance Mechanisms
Research is focused on the identification of plant salt tolerance determinants. Plant genes are isolated by functional selection as suppressors of salt-sensitive yeast mutants, as homologues of yeast genes involved in ion homeostasis or by interaction with plant or yeast tolerance determinants. כמוכן, Arabidopsis mutants are screened for genotypes with altered stress responsiveness. The functionality of stress tolerance determinants is being confirmed by expression in transgenic plants based on sufficiency for stress tolerance or suppression of stress-sensitive mutants. Arabidopsis GSK3/shaggy-like that complements yeast salt stress-sensitive mutants is induced by NaCl and abscisic acid. Plant Physiol Apr;119(4): GSK3/shaggy-like genes encode kinases involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain with a NaCl stress-sensitive phenotype, Stress-induced protein phosphatase2C is a negative regulator of a MAPK Previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of MAPK pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding - Genomic approaches High-throughput analysis systems are now replacing the classical gene-by-gene approaches in studies of gene expression and function. A genome-wide analysis of transcriptional responses to salt in organisms from yeast to higher plants. About 8% of all transcripts are responsive in every organism (500–3000 genes). In rice, the early response (in 1st h) to salt stress is critical for tolerance. It includes many transcripts required for signal transduction pathways and are more apparent in salt-tolerant varieties

A major gap in understanding salt toxicity is the nature of the targets at the cellular level
- The cell division cycle is one such target and the activity of the cyclin-dependent kinase (CDK) complex is decreased in salt-stressed Arabidopsis roots. - Another important target of salt toxicity seems to be RNA processing, because overexpression of serine-arginine-rich (SR) proteins involved in this phenomenon improves the salt tolerance of both yeast and Arabidopsis - In extreme salinity, plants must maintain high cytoplasmic K+/Na+ ratio and therefore must take up K+ efficiently in high [Na+] and be able to exclude or remove Na+ from the cytoplasm. - The approaches included characterization of mutants, determination of the expression patterns and expression of recombinant proteins in yeast and insect cells. The inward AKT1 is a major route of K+ uptake from soil by root epidermis. The outward SKOR at the root stele mediates xylem loading of K+. Highly selective channels of K+ over Na+ are unlikely to transport Na+ during salt stress. - One major pathway for Na+ uptake is blocked by external calcium and occurs via non-selective cation channels T-DNA-tagged lines derived from the sos3 mutant identified HKT1 transporter as suppressor - One common second messenger of diverse stresses, H2O2, activates AtANP1 (MAPKKK) cascade and AtMPK3,6 (two MAK kinases). This pathway represses auxin-inducible genes (GH3 and ER7) and induces stress defence genes (GST6 and HSP18). Truncation of the regulatory domain of AtANP1 creates a constitutively active kinase, which, upon expression in transgenic plants, improves the tolerance to multiple stresses such as cold, heat, drought and salinity. - ATHK1, a two-component histidine kinase homologous to the yeast osmosensor Sln1, which is able to complement the sln1 mutation. Sln1 is a negative regulator of the HOG1 MAP kinase pathway, which is counteracted by osmotic stress. Using the yeast system, a dominant negative ATHK1, was isolated which, upon expression in transgenic Arabidopsis, caused a constitutive stress response involving many genes and improved tolerance to drought, salt and cold but resulted in some growth retardation

Transcriptional cascades of low temperature and dehydration signal transduction
ABA-dependentTFs are shaded, while ABA independent are not. Small circles indicate posttranscriptional modification, such as phosphorylation. TF binding sites represented as rectangles at the bottom of the figure, with the promoters listed below. Dotted lines indicate possible regulation. Double arrow lines indicate possible cross talk.

Change in carbohydrates in response to salinity
Note that The accumulation of soluble carbohydrates in plants as a response to salinity or drought occurs despite a significant decrease in net CO2 assimilation rate In sunflower the salt tolerant lines had generally greater soluble sugars BUT in safflower there's NO correlation

Influence of salt stress on activity of plasma membrane H+-ATPase in some plant species

Changes in polyamines in plants species under salt stress

NahG plants are more tolerant to NaCl
What's the SA got to do with NaCl???

Na+ transport processes influencing Na+ tolerance in higher plants
Mitochondria peroxisomes Red arrows indicate Na+ movement, the minimization of which would increase tolerance; green arrows represent Na+ movements, the maximization of which would increase tolerance. The coloured shapes in the leaf represent chloroplasts (green), mitochondria (orange), peroxisomes (red) and endoplasmic reticulum (dark blue). Na+ transport processes into and out of these organelles is unknown. Vacuoles are represented by light blue shapes.

Factors affecting the energetics of Na+ efflux into the xylem
Assuming a 1 : 1 stoichiometry for Na+ : H+ exchange, then Na+/H+ antiporters will transport Na+ into the xylem, due to the large pH difference between the cytosol and xylem. However, if the xylem pH changes, or if the stoichiometry of the antiporter is different, then antiporters could act to pump Na+ out of the xylem solution. If the intracellular concentration of Na+ is much higher than the xylem concentration, and if xylem parenchyma cells are slightly depolarized at high NaCl, then efflux to the xylem can occur passively via ion channels (right)

Model of the the role of the salt overly sensitive (SOS) pathway in mediating salinity tolerance by controlling Na+ flux through SOS1. Like the calcineurin pathway in yeast, Ca2+ acts as a second messenger for the SOS pathway Arrows with black arrowheads represent Na+ movement through unidentified flux system(s). Arrows with white arrow heads represent an antiporter system From > T-DNA lines ofthe sos3 mutant, 2 null alleles of HKT1 transporter acted as suppressors of both the Na+ sensitivity and Na+ hyperaccumulation of the sos3 mutant. These results constitute the in vivo demonstration that HKT1 is an entry system for Na+.

Salt stress-regulating genes (see next slide)
SOS3 is a Ca2+BP that contains EF-hands and a myristoylation site in the N terminus. It has homology with yeast calcineurin subunit B and animal Ca2+ sensors. SOS2 is a Ser/Thr kinase similar to yeast sucrose nonfermenting (SNF1) kinase and the mammalian cAMP-activated PK SOS1 is a plasma membrane Na+/H+ antiporter resembling the mammalian NHE and bacterial NhaP exchangers SOS1 expression is upregulated by salt stress in plants but this upregulation is reduced by sos3 or sos2 mutations Genetic complementation studies Salt stress elicits rapid increase in free cytoplasmic Ca2+. SOS3, a myristoylated Ca2+BP, that can sense this calcium signal. SOS3 also recruits SOS2 to the plasma membrane, where the SOS3-SOS2 protein kinase complex phosphorylates SOS1 to stimulate its Na+/H+ antiport activity. Loss-of-function mutations in SOS3, SOS2, or SOS1 cause hypersensitivity to Na+

The SOS pathway functions in ion homeostasis under salt stress
High extracellular concentrations of salt elicit a rise in cytosolic Ca2+. The Ca2+ sensor SOS3 upon the perception of this signal interacts with and activates the protein kinase SOS2. Activated SOS2 then regulates the ion transporter activities or TFs to regulate ion homeostasis or gene expression. The SOS2 targets include the SOS1 Na+/H+ antiporter, the vacuolar Na+/H+ exchangers NHX,and the Na+/K+ transporter HKT1. Other targets include tonoplast ATPase and pyrophosphtases,water channels and K+ transporter

Activation of SOS2 protein kinase by SOS3 Ca 2+–binding protein
regulatory and catalytic domains of SOS2 interact, resulting in autoinhibition of the kinase SOS2 regulates the activity of ion transporters by myristoylation, SOS3 may also help to recruit SOS2 to specific membrane localization (not shown). B regulatory and catalytic domains of SOS2 interact, resulting in autoinhibition of the kinase A) If SOS3 is inactive, the kinase activity of SOS2 is inhibited by interaction between the C-terminus and the kinase domain through the conserved (FISL) motif. (B)Upon binding to Ca2+, SOS3 becomes active and then interacts with the FISL motif and releases its inhibition of SOS2 kinase activity. This also provides substrate accessibility to SOS2 kinase domain. Through protein phosphorylation,

 Signaling cascade involved in the development of salt tolerance in Arabidopsis. N-myristoylation of the Ca2+ binding protein SOS3 (salt overly sensitive 3) is required for a proper functioning of the SOS3/SOS2 protein kinase complex in planta. As indicated in the text, the SOS1 protein acts as a putative Na+/H+ antiporter and as a downstream effector of the SOS3/SOS2 complex. How myristoylation of SOS3 improves the function of the SOS3/SOS2 complex is as yet unknown. Myr represents the myristate moiety. Mammalian homologs of SOS3 and SOS2 and their activating signals are shown for comparison.

Salt tolerance and apoptosis are survival and death mechanisms that are indispensable for normal development and tissue homeostasis in both plants and mammals. The critical role of N-myristoylation in the regulation of these processes is a clear illustration of the conservation of essential regulatory principles during evolution.

The three aspects of salt tolerance in plants (homeostasis, detoxification and growth control) and the pathways that interconnect them Homeostasis is broken down into ionic and osmotic homeostasis. The SOS pathway mediates ionic homeostasis and Na+ tolerance. MAPK cascade (similar to the yeast HOG1) acts in osmotic homeostasis. The two primary stresses (ionic and osmotic) cause secondary oxidation stress. Lea proteins function in alleviation of damages. CBF/DREB TFs mediate stress protein expre caused by high salt concentrations, cold, drought or ABA. The ionic homeostasis, osmotic homeostasis and detoxification pathways are proposed to feed actively into cell division and expansion regulation to control plant growth.

signal transduction in Arabidopsis under salt-stress
8:200 (2003) It is unknown whether high Na+ is detected extracellularly or in the cytosol and Na+ sensors not found. Na+ stress induces cytosolic Ca2+ (a component in Na+ stress signaling?). Na+ influx --> toxicity, include non-selective cation channels and HKT1. SOS3 is a Ca2+ sensor homolog that activates SOS1. SOS1 mRNA accumulates under salt stress

Salt stress activates several protein kinase pathways
SIPKK and SIMKK are MAPK kinases that interact with SIPK and SIMK, respectively Na+ elicits a cytoplasmic Ca2+ signal that is perceived by the Ca2+-binding protein, SOS3 that interacts with and activates SOS2 protein kinase pathway that regulates multiple MAPK pathways. MAPK pathways are also activated by other signals such as SA, elicitors and wounding.

SOS in salt and ABA signaling
SOS3-Like Ca2+-Binding Protein Diagram showing that the SOS3-SOS2 signaling module functions in a salt stress-elicited Ca2+ signaling pathway, which mediates salt tolerance. Similarly, various SCaBP-PKS complexes have been implicated in Ca2+ signaling pathways in response to abscisic acid (ABA), sugar, high pH, or drought and cold stresses.

Protein phosphatase SOS2 also activates SOS1 and Ca2+/H+ (CAX1) exchangers on the vacuolar membrane. Protein phosphatase ABI2 interacts with SOS2 inactivating SOS2. The SOS pathway may down-regulate the activity of Na+ influx transporters (AtHKT1 and NCS).

PM and vacuole in salt tolerance
Salt stress induced Ca2+ signals are perceived by SOS3, which activates the SOS2 kinase. Activated SOS2 kinase phosphorylates the SOS1 The SOS3-SOS2 kinase complex may regulate Na+ compartmentation by activating NHX1, and also may restrict Na+ entry into the cytosol, e.g. by inhibiting the plasma membrane Na+ transporter HKT1 activity.

Phenotypes of los5 Mutant Plants
LOS5 is a molybdenum cofactor (MoCo) sulfurase that generates the sulfurylated form of MoCo, a cofactor of aldehyde oxidase that functions in the last step of ABA biosynthesis in plants wt los5-1 wt los5-1 wt los5-1 RD29A-LUC Luminescence (A) Morphology of wt (left) and los5-1 (right) (B) Luminescence of (A) after low-temperature treatment at 0°C for 48 hr. (C) Morphology of wt (left) and los5-1 (right) (D) Luminescence of (C) after treatment with 100 µM ABA for 4 hr. (E) Morphology of wild-type (left) and los5-1 (right) in 300 mM NaCl. (F) Luminescence of (E) after 5 hr of 300 mM NaCl treatment. (G) Quantitation of the luminescence intensities of wild-type and los5-1 plants in response to cold (0°C for 48 hr), ABA (100 µM for 4 hr), or NaCl (300 mM for 5 hr) treatment as shown in (B), (D), and (F). Also shown are data for untreated plants (control). (H) Low temperature dosage–response curves. Treatments at -5 or -10°C lasted for 3 hr, followed by incubation at room temperature for 2 hr. Treatment at other temperatures lasted for 48 hr before imaging. (I) NaCl dosage–response curve. Treatment time was 3 hr. The color scale at right shows the luminescence intensity from dark blue (lowest) to white (highest). Data in (G) to (I) represent means and ±SEs (n = 20). Open symbols, wild type (WT); closed symbols, los5-1. cold ABA 300mM NaCl Q u a n t i t a t i o n o f t h e l u m I n e s c e n c e Isolation of two allelic Arabidopsis mutants, los5-1 and los5-2 impaired in gene induction by cold and osmotic stresses

Freezing Sensitivity of los5-1 Plants
-7°C for 5 hr

Proline Accumulation and Osmotic Stress Sensitivity of los5-1 Mutant Plants
Drought sensitivity los5-1 plants are more sensitive to NaCl stress

LOS5 Regulates Cold– and Osmotic Stress–Responsive Genes through Distinct Mechanisms
RD29A-LUC Expression Cold responsiveness in los5-1 mutant is not rescued by application of ABA.

A Generic Pathway for the Transduction of Cold, Drought, and Salt Stress Signals in Plants.

ABA metabolism is regulated by osmotic stress at multiple steps

Major Types of Signaling for Plants during Cold, Drought, and Salt Stress
Type I signaling involves the generation of ROS scavenging enzymes and antioxidant compounds as well as osmolytes Type III signaling involves the SOS pathway which is specific to ionic stress

Osmotic homeostasis and ROS detoxification under salt stress
Osmotic homeostasis and ROS detoxification under salt stress. Ca2+ signals sensed by CDPKs are transduced through unknown signaling intermediates, which induce genes encoding LEA-like proteins ABA induced Ca2+ signals are perceived by SCaBPs, which activate PKS. The ABA signaling pathway upregulates osmolyte biosynthesis and LEA-like proteins under salt stress. Ca2+ signaling through CDPKs and SCaBPs is under negative control of Protein Phosphatase 2C (ABI 1/2). High osmolarity may be perceived by AtHK1, which presumably transduces the signal through a MAPK pathway. Salt stress and reactive oxygen species (ROS) activated MAPK (ANP1 & AtMEKK1 =MAPKKK; AtMEK1=MAPKK; AtMPK3, 4 & 6 = MAPK) cascade may regulate oxidativestress management (Broken arrows indicate unknown signaling intermediates).

Freezing and high-salt stress tolerance of the 35S-OsDREB1
PJ33;751

Phenotypes of the 35S:DREB plants in relation to wild-type plants (pBI121)
Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor Nature Biotechnology 17, 287 - 291 (1999) overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Freezing, drought, and high-salt stress tolerance of the transgenic plants
rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Pathways for the Activation of the LEA-Like Class of Stress-Responsive Genes with DRE/CRT and ABRE cis Elements The HOS1 locus negatively regulates cold signaling, presumably by targeting ICE or upstream signaling components for degradation Cold, drought, salt stress, and ABA can activate genes through stress-inducible transcription factors CBF/DREB1 and DREB2, and ABA-inducible bZIP TFs ABF/AREB An unidentified transcriptional activator, ICE (inducer of CBF expression), is indicated. IP3 is involved in the signaling, as revealed by genetic identification of the FRY1 locus, which negatively regulates IP3 levels and stress signaling

Engineered drought and freezing tolerance in transgenic B
Engineered drought and freezing tolerance in transgenic B. napus through constitutive expression of CBF1 A, Three-week-old plants were frozen at –6°C for 2 d and then let to recover for 2 d at 28°C before pictures were taken. B, Seven-week-old greenhouse grown plants were withheld water for 1 week and then rewatered for 2 weeks before picture was taken

osmotic stress regulation of early- and delayed-response genes
A) Model integrating stress sensing, activation of phospholipid signaling and MAPK cascade, and transcription cascade leading to expression of delayed-response genes. B) Examples of early-response genes encoding inducible transcription activators and their downstream delayed-response genes encoding stress tolerance effector proteins.

proposed functions of ion channels in ABA signaling and stomatal closing

Salt Tolerance Correlates with K+ Content but Not with Na+ Content in Seedlings
K+ content in whole seedlings. Na+ content in whole seedlings. Five-day-old seedlings on MS agar plates were transferred to media with or without 50 mM NaCl and allowed to grow for 48hr. Open bars, wt; black bars, sos1-1; stippled bars, sos2-1; striped bars, sos3-1 Relative root growth

K+ requirement of sos2 Optimal Growth of sos2 Requires Increased External K+ in the Culture Media.

Comparison of Salt Sensitivity among sos1, sos2, and sos1 sos2 double mutants
The data indicate that the two mutations are not additive and support the notion that the SOS genes function in a linear pathway. wt sos1 sos2 sos1+2

Salt stress-regulating genes
Mutants that have enhanced NaCl sensitivity, npct1, a gene encoding a Na+-dependent phosphate transporter–like protein. hkt1 mutations as suppressors of Na+ hypersensitivity by signal components downstream of SOS3, components of a parallel regulatory pathway(s), or other salt tolerance effectors regulated by stress signal pathways, or might be intragenic mutations in the sos3–1 allele. and K+ deficiency in sos3 mutants, implicating the AHKT1 protein in Na+ and K+ acquisition. The hkt1 suppressor mutant has a lower Na+ content, implying that AtHKT1 mediates Na+ uptake into plants. hkt1 also abrogate the growth inhibition of the sos3 mutant that is caused by K+ deficiency with low Ca2+. AtHKT1 is a Na+/K+ transporter that functions as a salt tolerance determinant that controls Na+ entry into plant roots hos15, was isolated as a hyperresponding luminescent mutant in transgenic plants expressing luciferase under the control of the Rd29A promoter. The hos15 mutant is hyperluminescent for cold, abscisic acid, and NaCl induction of the Rd29A promoter, but it is hypersensitive only to cold treatment. SOS1 activity is enhanced by pre-treatment with salt stress. AtHKT1 protein, a Na+/K+ transporter, is capable of mediating inward Na+ currents in Xenopus oocytes and K+ uptake in E. coli. HKT1 proteins are members of a superfamily of K+ transporters. For example, the Arabidopsis mutant uvs66 is affected in the perception of signals triggered by genotoxic treatments (UV light and DNA-damaging chemicals). The uvs66 mutant is also hypersensitive to ABA and salinity. NaCl sensitivity is specific to Na+ because the mutant is resistant to high KCl and hyper-osmolarity

H2O2- and ABA-activated ICa2+ currents are Na+ permeable in guard cells
(A) Whole-cell current recordings without ABA and with 50 ABA in the same guard cell bathed in 200 mM NaCl. (B) Average Na+ currents at -196 mV show that both ABA and H2O2 activated inward Na+ currents in Arabidopsis guard cells (H2O2, n = 7 cells; ABA, n = 6 cells) The EMBO Journal (2003) 22, 2623–2633,

Potential Pathways for Inositol 1,4,5-Trisphosphate (IP3) Degradation in Plants
FIERY1 inositol polyphosphate 1-phosphatase can hydrolyze Ins(1,4)P2 and Ins(1,3,4)P3. A potential pathway mediated by FIERY1 with direct hydrolysis of IP3 at the 1-position is also indicated (with a question mark). SHIP1: 5-phosphatase, inositol polyphosphate 5-phosphatase.

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