Abiotic stresses

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Lecture 21
Abiotic stresses Chilling and Freezing
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Chilling vs. Freezing

• Chilling injury
• Occurs at 10-15ºC on plants grown at warm
  temperatures (25-35ºC) ? tropical and subtropical
  species are susceptible to chilling (maize, bean,
  rice, tomato, cucumber, cotton).
• Growth is slowed
• Discoloration or lesions
• Foliage looks soggy, as if soaked
• in water
• - If roots are chilled, plants may wilt

Freezing injury Occurs at temperatures below
freezing point of water. Induction of tolerance
to freezing (as with chilling) requires a period
of acclimation at cold temperatures.
Citrus leaf
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Genetic adaptation to colder temperatures
associated with high altitude improves chilling
resistance
Survival at low temperature of seedlings of
different populations of tomato collected from
different altitudes in South America. Seeds
collected from wild tomato (Lycopersicon
hirsutum) and grown in the same greenhouse at
18-25ºC. All seedlings were then chilled for 7 d
at 0ºC and kept for 7 d in a warm growth room,
after which the number of survivors was counted.
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Membrane properties change in response to
chilling injury
Plants injured by chilling show inhibition of
photosynthesis slower carbohydrate
translocation lower respiration
rates inhibition of protein biosynthesis increas
ed degradation of existing proteins
Loss of membrane function during chilling
Solutes leak from leaves of chilling-sensitive
Passiflora maliformis (conch apple) floated on
water at 0ºC, but not of those from
chilling-resistant Passiflora caerulea
(passionflower). Loss of solutes to the water
reflects damage to the plasma membrane and
possibly also to the tonoplast. Inhibition of
photosynthesis and respiration reflects injury to
chloroplast and mitochondrial membrane.
Why are membranes affected by chilling?
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Plant membranes consist of a lipid bilayer
interspersed with proteins and sterols
Why are membranes affected by chilling?
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Membrane properties change in response to
chilling injury
In chilling-sensitive plants, the lipids in the
bilayer have a high percentage of saturated fatty
acid chains (have no double bonds!) ? tend to
solidify into a semi-crystalline state at a
temperature well above 0ºC. As membranes become
less fluid, their protein components can no
longer function normally ? inhibition of
H-ATPase activity, solute transport in and out
of cells, enzyme-dependent metabolism
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Membrane lipids from chilling-resistant plants
often have greater proportions of unsaturated
fatty acids than chilling-sensitive plants
During acclimation to cool temperatures the
activity of desaturase enzymes increases and the
proportions of unsaturated lipids rises ? lowers
the temperature at which the membrane lipids
begin a gradual phase change from fluid to
semi-crystalline and allows membranes to remain
fluid at lower temperatures. Desaturation of
fatty acids ? protection against damage from
chilling!
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Changes in relative levels of cis-unsaturated
molecular species of phosphatidylglycerol (PG) in
thylakoid membranes affect sensitivity to
chilling
Web topic 26.3
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Chilling-sensitive leaves exposed to high photon
fluxes and chilling temperatures are
photoinhibited, causing damage to the
photosynthetic machinery
Photoinhibition the inhibition of
photosynthesis by excess light
- comprises a complex set of
molecular processes
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Photoinhibition of photosynthesis under chilling
conditions
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Photoinhibition of photosynthesis under chilling
conditions
Ascorbate- Gluthatione Cycle
SOD Superoxide Dismutase
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Photoinhibition of photosynthesis during chilling
Xanthophyll cycle
(and others)
(Carotenoids SOD, ascorbate- glutathione cycle)
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Freezing stress Ice crystal formation and
protoplast dehydration kill cells
Fully hydrated, vegetative cells can retain
viability if they are cooled very quickly to
avoid formation of large, slow-growing ice
crystals that would puncture and destroy
subcellular structures. Ice crystals forming
during very rapid freezing are too small to cause
mechanical damage. Conversely, rapid warming of
frozen tissue is required to prevent growth of
small ice crystals into crystals of a damaging
size, or to prevent loss of water vapor by
sublimation (both happen -100 to -10ºC). Under
natural conditions cooling of intact plant
organs is never fast enough ? - ice forms
first within the intercelluar spaces and in xylem
vessels (not lethal to hardy plants and tissue
recovers fully if warmed) - exposure to
freezing for extended periods ? growth of
extracellular ice crystals results in
movement of liquid water from protoplast to
extracellular ice, causing excessive dehydration
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Ice crystal formation and protoplast dehydration
kill cells
During rapid freezing, protoplast, including
vacuole supercool, i.e. the cellular water
remains liquid even at temperatures several
degrees below its theoretical freezing
point. Several hundred molecules needed for ice
crystal to begin forming ice nucleation
(dependent on properties of involved surface
some large polysaccharides and proteins
facilitate ice crystal formation ice
nucleators)
Temperature of parenchyma cells in cucumber
(Cucumis sativus) fruit during freezing. The
temperature was recorded with an electronic
device, a thermistor, inserted into a 5 20 mm
cylinder of tissue and immersed in a coolant at
5.8C. (AB) Supercooling. (BC) Release of heat
during freezing in cell walls and intercellular
spaces. (CD) Supercooling. (DE) Small heat
spikes released during intracellular freezing of
individual protoplasts.
Web Topic 26.4
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Freezing stress Ice crystal formation and
protoplast dehydration kill cells
Model illustrating the freezing process in plant
cells. Ice nucleation occurs at a characteristic
sub-zero temperature. This creates a gradient in
the water potential between the extracellular and
intracellular compartments that draws water out
of the cytoplasm. The cell collapses as the ice
expands. In non-acclimated tissue, ice formation
is lethal. In acclimated tissue, de-hydration is
fully reversible to a characteristic threshold.
Cell death is apparent as solute leakage across
the plasma membrane and loss of turgor.
Limitation of ice formation contributes to
freezing tolerance!
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How is ice formation limited in the tissue?
Once the ice forms, its further growth and
propagation through the tissue is restricted by
numerous factors including cell wall
modifications, arabinoxylans and antifreeze
proteins (AFP). - Identified from a wide
variety of sources including polar fish, insects,
bacteria, and plants. - Adsorb onto the ice
crystals and modify their growth. - Depress the
freezing point of a solution without affecting
its melting temperature. - Considerable
recent progress has been made in studying AFP in
leaves of winter rye and carrot. -
Localized in the epidermis and in cells
surrounding intercellular spaces in
cold-acclimated rye.
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Transgenic Arabidopsis expressing an insect AFP
show freezing at significantly lower temperatures
than WT
Dendroides canadensis (fire-colored beetle)
antifreeze protein (DAFP-1) sequence showing the
location of disulfide bridges. Numbers identify
cysteines involved in the various disulfide
bridges.
Temperature at which initial freezing occurred in
WT and various transgenic Arabidopsis lines.
After being placed in the freezing chamber,
plants were equilibrated to 0ºC and then cooled
at a rate of 5ºC/h (0.083ºC/min).
The transgenic plants did not survive freezing
any better than wild-type plants. However, they
did show freezing at significantly lower
temperatures than wild-type plants. 
Huang et al. 2002 Plant Mol Biol 50 333
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Some bacteria that live on the leaf surface
increase frost damage
Frost sensitive plants are injured as a
consequence of ice formation between -2 and
-5C. Frost usually occurs on plants which have
not acclimated or which are unable to acclimate
to freezing stress, and thus are sensitive to any
ice formation
Pseudomonas
syringae
Erwinia herbicola
Bacteria act as nucelators at 2ºC.
Presence of these bacteria cause ice formation at
much higher temperatures than observed on sterile
plants. Environmental factors that favor growth
at these ice nucleation active bacteria make the
plants more susceptible to frost injury. The gene
responsible for ice nucleation activity has been
isolated and cloned from Pseudomonas syringae.
The gene encodes a 150,000 kD protein that is
associated with the outer membrane of the
bacteria.
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ABA and protein synthesis are involved in
acclimation to freezing

• ABA has a role in induction of freezing
  tolerance.
• Plants develop freezing tolerance at
  non-acclimating temperatures when
• treated with exogenous ABA.
• ABA-insensitive mutants (abi1) or ABA-deficient
  mutants (aba1) are
• unable to undergo low temperature acclimation
  to freezing.
• However, not all genes induced by low
  temperature are ABA-dependent.
• Exogenous ABA cannot confer the same freezing
  acclimation that
• exposure to low temperature does.
• - Minimum of several days (tomato needs 15 d) of
  exposure to cool temperatures is required for
  freezing resistance to be fully induced.
• - When re-warmed, plants lose their freezing
  tolerance rapidly
• (can become susceptible to freezing once again
  within 24hrs).

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Numerous genes are induced during cold acclimation

• More than 100 genes are up-regulated by
  cold-stress
• Heat shock proteins act as molecular
  chaperones
• up-regulated under cold and heat stress
• stabilize protein structure during cold and
  heat stress
• Antifreeze proteins many encode endochitinases
  and endoglucanases,
• which are induced upon infection of pathogens
  (pathogenesis-related
• proteins)
• Proteins involved in synthesis of osmolytes
• Proteins for membrane stabilization (LEA
  proteins)

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A transcription factor regulates cold-induced
gene expression

• Many cold stress-induced genes are activated by
  transcriptional activators
• C-repeat binding factors (CBF1, CBF2, CBF3)
• Dehydration Response Element Binding (DREB1b,
  DREB1c, DREB1a)
• Bind to CRT/DRE elements (C-repeat/dehydration-res
  ponsive, ABA-independent sequence elements)
• CBF involved in the coordinate transcriptional
  response of numerous cold and osmotic
  stress-regulated genes, all of which contain the
  CRT/DRE elements in their promoters
• CBF1 is specifically induced by cold stress and
  NOT by osmotic or salinity stress (DREB2 induced
  by osmotic and salinity stress and NOT by cold)
• Expression of CBF is controlled by separate
  transcription factor ?
• ICE (INDUCER OF CBF EXPRESSION)
• - Not induced by cold presumably activated
  posttranscriptionally by associated protein,
  permitting activation of CBF
• Transgenic plants constitutively expressing CBF1
  have more cold-up-regulated
• gene transcripts and are more cold-tolerant
  than wild type

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Pathways for the activation of stress-responsive
genes upon cold and dehydration
Zhang et al. 2004 Plant Physiol 135 615-621
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Engineered drought and freezing tolerance in
transgenic Brassica napus through constitutive
expression of CBF1
Zhang et al. 2004 Plant Physiol 135 615-621