Proof of Concept
We explored three strategies to protect apricot trees from frost damage. The first strategy is designed to protect the crops from ice crystals, arisen due to low temperatures, whereas the second and third strategies are methods to diminish the damage done by Pseudomonas syringae pv. syringae (PSS), a pathogen whose ice nucleation proteins (INPs) cause the increase of the temperature at which freezing occurs.
To demonstrate the feasibility of our project we came up with proofs of concept for two of our strategies we have:
Antifreeze Proteins
One of our strategies is using antifreeze proteins (AFPs) to protect apricot trees against frost damage. AFPs can bind to ice and lower the freezing temperature due to their characteristics to change their thermal hysteresis corresponding to the difference between melting and freezing temperatures (TH), thus reducing frost damage done to plants. We developed two assays. The first assay proves that AFPs we produced change the TH of a solution and that each tested AFP differs from each other. The second one shows that AFPs can reduce frost damage to plants. The following sections will describe the assays in detail.
ISF Assay
The first assay is the “I Said Freeze” (ISF) assay, where we measured the thermal hysteresis (TH) of our solution with our AFPs. When AFPs were added to a solution, they affected the TH in correlation with their concentrations. The TH is therefore a measurement that is specific to each AFP’s activity - measuring it allows us to verify that we produced the desired protein and that it is functional.
To measure the TH, we designed a device that is able to precisely cool and heat up to a certain temperature, allowing us to freeze and thaw our samples. In parallel, we visualised the freezing and melting of our drops of solution through a microscope. This setup allowed us to pinpoint the freezing and melting temperatures of our protein solutions at varying concentrations, and therefore the TH of our proteins at these concentrations. To find out more about the various measurements we did, click here.
Connected scatter plot graph showing the mean of biological triplicate +/- standard deviation. Thermal hysteresis is measured with two different AFPs, RiAFP and FfIBP, at different concentrations (µM).
The TH measurements we obtained for FfIBP and RiAFP prove that our AFPs are active. Next, we wanted to demonstrate the ability of AFPs to lower the freezing temperatures in a solution. For this experiment, we compared drops of buffer containing each of our AFPs to an untreated one as a control. This allowed us to compare the freezing temperatures between AFP solutions and the buffer. From this comparison, we demonstrated that the presence of our AFPs in our solution significantly decreases freezing temperature, proving that they are functional.
mean of the freezing temperature of FfIBP [200µM], RiAFP[50µM] and the control with their error plot representing the confidence interval. We used the most concentrated solution we had for every AFPs we used. The control has the same buffer as in the other drop but without any AFP or protein.
In conclusion, the ISF assay allowed us to show that our AFPs work as expected - they reduced freezing temperature and changed the TH of the solution.
FDT
The second assay we performed is the “Frost Damage Treatment” (FDT) assay, in which we measured the percentage of frost damage on Arabidopsis thaliana plants that were treated in different solutions.
To do so, we completely immersed two plants per solution and placed them in a climatic chamber at -5 ºC overnight. The next day, we stained the plants in order to measure the damage of the leaves caused by the freeze. The stain we used was Trypan blue, a dye that only enters dead pierced cells and colors them. Since AFPs protected our plants against frost, we expected to see less blue cells in leaves with AFP treatment as opposed to those without treatment, used as a control.
Leaves dyed with Trypan blue showing frost damage.
Left: a leaf previously immersed and frozen in a buffer without AFPs.
Right: a leaf previously immersed and frozen in a buffer with a mix of AFPs (FfIBP + RiAFP).
To measure the quantity of blue-stained cells in a leaf, we created a software, VISION, that calculates all the blue areas, which indicate damage, and divides this number by the total area of the leaf. With this information, the software efficiently determines the fraction of damage on the leaf. We can then numerically compare the frost damage done to a leaf treated with AFPs to one treated with the control buffer.
Results we obtained show that AFPs can reduce the damage on leaves by up to 12 times (Fig. 5). This proves that our AFPs efficiently reduce frost damage on plants. For more information about these measurements, click here.
The graph shows mean +/- standard deviation. Percentage of Damage on Arabidopsis thaliana leaves measured after overnight incubation in a climatic chamber at -5ºC. Plants were immersed in phosphate-buffered saline (PBS) liquid solution supplemented with two different AFPs: FfIBP [200 µM] (n=13) and RiAFP [50 µM] (n=17) and a mix of them (RiAFP + FfIBP) (n=15) at a concentration of 25 µM and 100 µM, respectively. These solutions were compared with the control in which the plants were immersed in the PBS buffer without any AFP (buffer) (n=17).
The damage ratio is significantly different between the buffer and all AFPs. ANOVA, * significant at p<0.05.Damage ratio between the different AFPs are not significant.
In conclusion, the FDT assay measures the area damaged by frost on the plant dyed with Trypan blue using our software VISION.
To summarize, we have demonstrated that the AFPs we chose (FfIBP and RiAFP) are able to lower freezing temperatures, differ in their TH and protect plants efficiently.
Tailocins
One of our strategies is to use tailocins to specifically kill Pseudomonas syringae pv. syringae that cause the increase of freezing temperatures in water through endogenous proteins INPs. Tailocins are phage-derived bacteriocins that specifically kill a narrow range of bacterial strains. To prove that we could decrease the freezing temperature of water containing PSS by treating the strains with tailocins, we made a new assay called “versus”. In this assay, we treated a water drop with the pathogens PSS and tailocins and compared the freezing temperatures of the treated drop with one containing only the pathogens. Then, we placed them side by side on our device FROZONE, a nanoliter osmometer that can precisely set and control the temperature of a copper plate. We set the setpoint of FROZONE at -15 ºC and observed which drop was freezing first. If tailocins reduced the ability of PSS to increase freezing temperatures of water, we should see a difference for the freezing time between the treated drop and the control.
Graph shows mean of biological triplicate +/- standard deviation. Time measured before observed freezing of NYB liquid solution supplemented with Pseudomonas syringae pv. syringae or PSS and tailocin proteins (Tailocin+PSS). Times before freezing are significantly different. T-test, * significant at p<0.05, p=0.0114.
We indeed observed that tailocins increased the freezing time around two-fold and the difference between infected (PSS with Tailocins) vs non-infected (only PSS) is statistically significant. This result proves that tailocins we produced can reduce the pathogenic effect of PSS and therefore reduce frost damage.
References
- Group FM. Staining of Arabidopsis thaliana leaves with trypan blue and aniline blue. 2010;(314):1–2.
- II H, M R, AG H. Mechanisms of frost resistance in Arabidopsis thaliana. Planta [Internet]. 2018 Oct 1 [cited 2021 Oct 20];248(4):827–35. Available from: https://pubmed.ncbi.nlm.nih.gov/29936546/