
Antifree proteins (AFPs) have been
isolated from several organisms.
› Fish, insects, plants, bacteria

Bind to ice crystals, inhibit growth, lower
freezing point and not melting point.

Thermal hysteresis activity (THA)difference between melting point and
nonequilibrium freezing point.

Used as an indicator of AFPs activity, so
AFPs often referred to as thermal
hysteresis proteins (THPs).

Varies among species: Insects (3-6 ºC),
Fish (0.7-1.5 ºC), Plants (0.2-0.5 ºC)

AFP-producing insects: goal is to avoid
freezing, cannot survive if body fluids
actually freeze.

AFPs lower freezing point of hemolymph
and gut fluid to prevent freezing from the
external ice across the body surface.

Can achieve higher crop yields by
improving freezing tolerance of plants.

Therefore, want to express AFPs in frostsusceptible crops to increase their cold
tolerance.

Some of the most effective ATPs found in
insects.

Want to test the MpAFP149 gene,
isolated from the beetle Microdera
puntipennis dzungarica,in its ability to
increase cold-tolerance of transgenic
tobacco plants and protect them from
freezing damage.
363 bp with a signal peptide sequence
 Transcript encoding 98 amino acids of
mature peptide is 68.37% homologous
with published AFP from Tenebrio molitor
(Tm)
 Tm expressed successfully in E. coli; high
activity at protecting bacteria at low
temperature, linearly correlated with AFP
concentration.

Confirm expression of MpAFP149 in
plants and visualize sub-cellular
localization
 MpAFP149 gene with 35S promoter fused
with green fluorescent protein (GFP) in
plasmid pCAMBIA1302-GFP
 Introduced into onion epidermal cells via
particle bombardment


MpAFP149 gene with signal peptide
sequence obtained by PCR.
Gene construct
CaMV35S-MpAFP149-Nos inserted into
plasmid pCAMBIA1302 with HindIII and
EcoRI to form expression vector
pCAMBIA1302-MpAFP149


Expression vector pCAMBIA1302MpAFP149 transferred into competent
Agrobacterium cells (EHA105 strain)

DNA extracted from kanamycin-resistant
surviving Agrobacterium colonies

PCR to confirm presence of MpAFP149
transgene

1-2 in. young tobacco leaf discs infected
with EHA105 Agrobacterium containing
pCAMBIA1301-MpAFP149.
Cultivated in the dark at 28 ºC for 2 days.
 Leaf discs transferred to generation
medium supplemented with hygromycin.
 T0 plants allowed to grow and flower
and set seeds in a growth chamber.


Allowed to grow 15 weeks in green
house before harvesting seed capsules

T0 and Wild-type seeds sterilized by
soaking in 1:9 (v/v) 30% bleach:ethanol

Rinsed 5 times with ethanol and left
overnight to volatize ethanol

T0 seeds germinated on plates with
hygromycin to select for seedlings
carrying HPTII gene

Transplanted into pots to full growth at
25 ºC

Extracted genomic DNA and performed
PCR to identify MpAFP149 gene

RNA isolated from plant leaves and
reverse transcription carried out

RT-PCR products run through agarose gel
electrophoresis to check MpAFP140
transcription

Wild-type leaves and transgenic
tobacco leaves

Polyclonal antibody raised in mouse
against MpAFP149 protein

Immunogold labeling (Antibodies
conjugated to gold particles)

Extracted apoplastic proteins from
leaves and separated by sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)

Western blot with antibody against
MpAFP149 protein

After growing for one month, three
transgenic and three wild-type plants of
similar growth states and no visible
phenotypical differences were chosen
to undergo cold treatment, measure
electrolyte leakage, and
Malondialdehyde (MDA) content.

Set temperature in freezing chamber to -1
ºC for 0, 24, 48, and 72 hours and observed
phentypes.
In addition, leaf samples from each group
were washed with deionized water and
then immersed in deionized water.
 After vacuum infiltration, the electric
conductivity of supernatant was detected.


MDA- natural occuring reactive species
that is a marker for oxidative stress

The level of MDA at -1 ºC was
determined to analyze the comparative
rate of lipid peroxidation.
The localization of MpAFP149 was
determined by expressing
MpAFP149:GFP construct plasmid in
onion epidermal cells.
 For the control, fluorescence was seen
throughout the entire cell and for the
transformed cells it was solely in the
apoplast (see Figure next slide).

35S-MpAFP149-NOS vector transformed
into tobacco using Agrobacteriummediated gene transfer.
 Screened by hygromycin and tested for
the presence of the vector by PCR.
 Two samples, T0-5 and T0-39 showed
higher transcript level by RT-PCR.
 These two lines were chosen for detailed
analysis.

Immunogold labeling approach used to
determine if MpAFP149 protein was
expressed and where it localized in
transgenic tobacco.
 Showed that MpAFP149 protein
accumulated in outer layers of cell wall
in transgenic plant, but absent in control
tobacco plant


Western blot for apoplastic proteins
showed expected protein band of 10.2
kDa, indicating that mature peptide
protein MpAFP149 synthesized in
transgenic tobacco.

When exposed for 1 day, both
transgenic and wild-type tobacco plants
only exhibited moderate dehydration.

When exposed for 2 and 3 days, most
leaves of wild-type were frozen but
transgenic tobacco only exhibited
dehydration of a few older leaves near
the plant base.

After returning to room temperature,
MpAFP149 plants overcame dehydration
and recovered completely.

Wild-type displayed permanent
damage.

Transgenic line displayed improved cold
tolerance and enhanced recovery
Low temperatures disrupted semipermeability of tobacco
cytomembranes
 Effusion of electrolytes resulted in
increased electrical activity of tissues
 Over time, ion leakage difference
increased between control and
transgenic tobacco.


Increase of MDA parallels the increase in
conductivity/ion leakage, one does not
cause the other.

Wild-type plants suffered higher
oxidative lipid injury than transgenic
plants; correlated to increases in ion
leakage and MDA content.

Transgenic tobacco plants expressing
MpAFP149 protein with the signal
peptide showed improved tolerance to
cold and an enhanced recovery.

MpAFP149 may be used as a candiate
for the improvement of frost-resistant
crops.

Wang, Y., Qiu, L., Dai, C., Wang, J., Luo,
J., Zhang, F., & Ma, J. (2008) Expression of
insect (Microdera puntipennis
dzungarica) antifreeze protein
MpAFP149 confers the cold tolerance to
transgenic tobacco. Plant Cell Rep 27:
1349-1358.