2.  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.

3.  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)

4.  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.

5.  Can achieve higher crop yields by improving freezing tolerance of plants.  Therefore, want to express AFPs in frost- susceptible crops to increase their cold tolerance.

6.  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.

7.  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.

8.  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

9.  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

11.  Expression vector pCAMBIA1302- MpAFP149 transferred into competent Agrobacterium cells (EHA105 strain)  DNA extracted from kanamycin-resistant surviving Agrobacterium colonies  PCR to confirm presence of MpAFP149 transgene

12.  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.

13.  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

14.  T0 seeds germinated on plates with hygromycin to select for seedlings carrying HPTII gene  Transplanted into pots to full growth at 25 ºC

15.  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

16.  Wild-type leaves and transgenic tobacco leaves  Polyclonal antibody raised in mouse against MpAFP149 protein  Immunogold labeling (Antibodies conjugated to gold particles)

17.  Extracted apoplastic proteins from leaves and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)  Western blot with antibody against MpAFP149 protein

18.  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.

19.  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.

20.  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.

21.  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).

23.  35S-MpAFP149-NOS vector transformed into tobacco using Agrobacterium- mediated 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.

25.  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

27.  Western blot for apoplastic proteins showed expected protein band of 10.2 kDa, indicating that mature peptide protein MpAFP149 synthesized in transgenic tobacco.

29.  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.

30.  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

32.  Low temperatures disrupted semi- permeability of tobacco cytomembranes  Effusion of electrolytes resulted in increased electrical activity of tissues  Over time, ion leakage difference increased between control and transgenic tobacco.

33.  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.

35.  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.

36.  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.