Transcript Isolation of salt sensitive mutants

Salinity Impact on Crop Production Worldwide
World Land Surface Area
Salt affected
150 x 10 6 km 2
9 x 10 6 km 2 (6%)
Cultivated Land
*Salt affected
15 x 10 6 km 2
2 x 10 6 km 2 (13%)
Irrigated Land
*Salt affected
2.4 x 10 6 km 2
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
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tolerate
high salinity.
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
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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, 1718-1737.
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Sensitivity to salt occurs during all plant growth stages
germination
NaCl inhibits both 1) germination and 2) growth
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Resistance to drought and salt stresses
by neutrally charged osmolytes
Osmolytes
Heat shock proteins
LEA
Compatible solutes protect
the hydration shell
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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
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Salt stress
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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
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If the halophytes’ and glycophytes’
enzymes are equally sensitive to
NaCl, why are the plants
differentially sensitive to NaCl?
Salt Stress Effects on Plants
Primary
Water deficit
Ion disequilibrium, NaCl is the predominant salt:
Na+ reduces K+ acquisition resulting in K+ deficiency
Secondary
Reduced cell expansion
Reduced assimilate production
Reduced membrane function
Decreased cytosolic metabolism
Production of reactive oxygen intermediates (ROSs)
Etc.
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-
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
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Selective ion uptake and differential ion
compartmentalization are main features that explain salt
tolerance disparity between glycophytes and halophytes
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(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
NaCl induces cytological hallmarks of
programmed cell death in the wild-type yeast
Bc2-2 protects
Nuclear fragmentation also
IN PLANTS
Nuclear fragmentation (1 h)
normal mitochondrion
abnormal mitochondrion
d) Nuclear fragmentation;
e) vacuolation;
f) coalescence of vacuolar and
nuclear membranes;
g) cell lysis.
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Hamilton,E & Heckathorn, S (2001)
Plant Physiol. 126, 1266-1274.
IN PLANTS
Mitochondrial adaptations to NaCl.
Complex I is Protected by AntiOxidants 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
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
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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 nonvacuolated 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…
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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. 2000 ; Schachtman and Liu, 1999 ).
• 3) Survey genes whose expression is regulated by salt
stress (Zhu et al., 1997 ; Xiong and Zhu, 2001 ; Shinozaki and YamaguchiShinozaki, 1997 ; Ingram and Bartels, 1996 ; Bray, 1997 ; Bohnert et al., 1995 ).
This research is accelerated by using microarrays
al., 2001 ; Kawasaki et al., 2001 ; Bohnert et al., 2001 ).
(Seki et
• 4) Mutational analysis of salt tolerance determinants
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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
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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
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salt cress
Unlike Arabidopsis leaf morphology, salt cress displays succulentlike 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 ;
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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
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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.
With increased concentration of NaCl in the medium, Na+ ↑ whereas K+
content in seedlings transferred for 3d to high NaCl
↓.
Analysis of ion
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, 1943-1945
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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
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reduced growth and vigor under salt stress.
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
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Na+ UPTAKE/EXTRUSION IN THE PLANT CELL
Plasma Membrane
PPi
Na+
K+
Na+
H+
H+
High-affinity K+
transporters
V-PPase
H+
Na+
Na+/H+ antiport
Vacuole
Na+
Na+
K+
Tonoplast
V-ATPase
K+/Na+ selectiveVICs
H+ ATP
K+/Na+ ratio
H+
ATP
P-ATPase
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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
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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 )
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Sodium Fluxes through Nonselective Cation Channels in the
Plasma Membrane of protoplasts from Arabidopsis Roots
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, 27
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
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pyrophosphatase, ∆pH
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+
K+)(Na
K +
+)
H
Na+/H
+
K
+
c
p
*-scavenging
m
t
per
ox
Plasma
-120 to -200
AT
Membra
H P mV
AT
ne
PH +
+ PP
Hi
Ca2
+
+
polyols
proline pH
Na
7.5
++
betaine
Tonopla
st
NaCl↑
pH
Na+trehalose
ClCa2+
+205.5
to +50
H
mV
+
Na
H
+
Cl
Cl
Ca2
+
pH 5.5
-
-
Ca2
+
Cl
H +
+
H2
O
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
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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.
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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
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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
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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, 2113-2122
(A)
CAX1-expressing lines
(B)
CAX1-expressing lines after several
weeks in the greenhouse.
(C)
& (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.
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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,
34 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;19:967-973
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.
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(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.cerevisiae
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
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Mol. Cell. Biol. 17, 1289-1297
The yeast HOG1 signal transduction
pathway contains two independent
osmosensors. The first is a twocomponent 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
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
•
•
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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
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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
High39NaCl 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
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.
40 Fus3 and Hog1 are called MAPKs, Ste7 and Pbs2 are MAPKKs, and Ste11 is a MAPKKK.
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 lefthanded 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
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Activation of protein kinases by
hyperosmotic stress
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Osmotic stress-activated protein kinases in plants
MAPKs are induced by osmotic stress (salt and drought) stress
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
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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
The SLN1 branch
of the HOG
pathway is
stimulated by
turgor reduction
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
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.
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
44 Ssk2 and Ssk22. In contrast, another TM protein (Sho1) serves as a facilitator
kinases
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
CDPK, calmodulin-like domain protein kinase; DAG, diacylglycerol; DGPP, diacylglycerol pyrophosphate; IP3, inositol 1,4,5-trisphosphate; L-PA, lyso45
PA; MAPK, mitogen-activated protein kinase; PA, phosphatidic acid; PI3K, phosphoinositide 3-kinase; PI(3,5)P , phosphatidylinositol 3,52
bisphosphate; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D.
Algorithm for discovering stress tolerance determinants
46
Comparison of Salt Sensitivity in sos mutants on
Vertical Plates by Using the Root-Bending Assay
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 SOS3
47
Zhu Plant Phys 2000
Sensitivity of salt sensitive mutants to other salts
sos3 but not sos1 is fixed by high Ca
48
WT, sos1, and sos3 mutants were exposed to highsalt (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 Nmyristoylation signature sequence
49
Plant Cell, Vol. 12, 1667-1678
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
(A)Plants on 20 mM K +.
(B)Plants on 200 nM K*.
50
The K +content in the wild
type did not decrease to
<3%of the dry
weight,while in sos1 it decreased to ~1%, indicating
that K +deficiency occurs
in NaCI-treated sos1
plants.
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+
51
Sensitivity of sos2 and sos3 seedlings to Salts
sos3
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
52
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.
53
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.
54
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+
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
55
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, ABAdependent 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
56
Functional demarcation of salt and
drought stress signaling pathways.
Plants rarely experience stress from a single environmental source
57
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
58
mutations suppress the
hkt1-1 and hkt1-2
NaCl hypersensitive phenotypes of sos3-1
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
59
entry system for Na+.
AtHKT1 is a salt tolerance determinant that controls Na+
entry into plant roots (PNAS 98 (24): 14150-14155 NOV 20 2001)
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.
60
Drought-and salt-tolerant plants by
overexpression of the AVP1 H1-pump
WT AVP1-1 AVP1-2
after rewatering
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
61
toxic
effects.
Ion Homeostasis Transport Determinants
-120 to -180 mV
+50 mV
>500 mM Na+/Cl-
<100 mM Na+/Cl-
Plasma membrane:
500 mM Na+/Cl-
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
62
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
vacuolar Na+/H+ antiporter gene
AtNHX1, 2 and 5 (post-transcriptional?)
[Na+]ext↑ → [Ca2+]cyt↑ → SOS3 → SOS2
Negative regulation: AtHKT1
Na+ transporter that controls Na+ entry into roots
[Ca2+]ext blocks Na+ uptake through NSCC
Non-selective calcium channel
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
63
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
64
exerts its malicious effect by disruption of membrane integrity and solute leakage.
Plant Drought and Salt Stress Tolerance Mechanisms
http://www.nature.com/cgi-taf/DynaPage.taf?file=/embor/journal/v3/n2/full/embor229.html
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. 1999 Apr;119(4):1527-34
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
65
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 nonselective 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
66 many genes and improved tolerance to drought, salt and cold but resulted in some growth
involving
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
67
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
68
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
69
Changes in polyamines in plants species
under salt stress
70
NahG plants are more tolerant to NaCl
What's the SA got to do with NaCl???
71
Na+ transport processes influencing Na+
tolerance in higher plants
Mitochondria
peroxisomes
72
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
73
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.
Arrows with black
arrowheads
represent Na+
movement through
unidentified flux
system(s). Arrows
with white arrow
heads represent an
antiporter system
74
Like the calcineurin pathway in
yeast, Ca2+ acts as a second
messenger for the SOS pathway
From >65 000 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
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
SOS3,75SOS2, 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
76
Activation of SOS2 protein kinase by
SOS3 Ca 2+–binding protein
A
regulatory and catalytic domains of
SOS2 interact, resulting in autoinhibition
of the kinase
B
SOS2 regulates the activity
of ion transporters by
myristoylation, SOS3 may
also help to recruit SOS2 to
specific membrane
localization (not shown).
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
77
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
78 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
79
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
80
expansion regulation to control plant growth.
signal transduction in Arabidopsis under salt-stress
8:200 (2003)
81
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
kinase
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
82 activated by other signals such as SA, elicitors and wounding.
SOS in salt and ABA signaling
SOS3-Like Ca2+-Binding Protein
83
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
84
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
85
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
wt los5-1
wt los5-1
wt
los5-1
LOS5 is a molybdenum
cofactor (MoCo) sulfurase
that generates the
sulfurylated form of
aldehyde oxidase that
functions in the last step
Luminescence
RD29A-LUC
MoCo, a cofactor of
of ABA
in plants
cold
ABA
300mM
NaCl
Quantitation of t h e lumInescence
Isolation
of two allelic Arabidopsis mutants, los5-1 and los5-2
86
impaired in gene induction by cold and osmotic stresses
biosynthesis
Freezing Sensitivity of los5-1 Plants
87
-7°C for 5 hr
Proline Accumulation and Osmotic Stress
Sensitivity of los5-1 Mutant Plants
Drought sensitivity
88
los5-1 plants are more sensitive to NaCl stress
LOS5 Regulates Cold– and Osmotic Stress–
Responsive Genes through Distinct Mechanisms
RD29A-LUC Expression
89
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.
90
ABA metabolism is regulated by osmotic
stress at multiple steps
91
92
Major Types of Signaling for Plants
during Cold, Drought, and Salt Stress
Type I signaling involves the generation of
93 scavenging enzymes and antioxidant
ROS
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.
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
94
& 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
95
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 stressinducible transcription factor
Nature Biotechnology 17, 287 - 291 (1999)
96
Freezing, drought, and high-salt stress
tolerance of the transgenic plants
97
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,
98 negatively regulates IP3 levels and stress signaling
which
Engineered drought and freezing tolerance in transgenic B. napus
through constitutive expression of CBF1
99
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.
100
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
101
Salt Tolerance Correlates with K+ Content
but Not with Na+ Content in Seedlings
K+ content in whole seedlings.
Na+ content in whole seedlings.
Relative root growth
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
102
K+ requirement of sos2
Optimal Growth of sos2 Requires Increased External K+ in the Culture
Media.
103
Comparison of Salt Sensitivity among
sos1, sos2, and sos1 sos2 double mutants
wt sos1 sos2 sos1+2
104
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.
105
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;
106 ABA, n = 6 cells)
The EMBO Journal (2003) 22, 2623–2633,
Potential Pathways for Inositol 1,4,5Trisphosphate (IP3) Degradation in Plants
107
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.