Protective compounds choices against frost and heat

Overview

Once the different methods of Softer Shock application on the leaf and the choice of the expression host have been explored, it is now the time to think of how this latter will perform its protective action at the plant surface. Our core strategy lies in the thermo-responsiveness of our micro-organism, providing a dual response against extreme temperatures. Specific protectants will be synthesized according to meteorological events, and protect the plan from these eventual abiotic stresses.

What happens at the plant level in case of frost or heat episodes? Which compounds could prevent those deleterious effects?

During our investigations, we looked through diverse research axis and summarize here the main compounds that appeared the most promising for us.

We deeply thank Nicolas Aveline, Camille Lenoir, Christian Huyghe, Guillaume Charrier, Ido Braslavsky and Maya Bar Dolev for their valuable pieces of advice. Through several interviews, these experts helped us better understand the complex physiological mechanisms of heat and frost damage on plants. They also guided our choices concerning our different protective compound candidates. You can find the complete interviews in written form below:

Cold Protection

Below 0°C, freezing has several negative effects on grapevines as well as other plants. Spring frost episodes are particularly harmful, as the grapevine has already restarted its growth and crucial organs (buds and leaves) under formation can be easily destroyed¹. These events are characterized by sudden temperature drops, often happening at night, and can result in severe production losses as well as an alteration of fruit characteristics of the following harvest ².

The mechanism of ice crystal formation, called nucleation, is one of the main factors causing diverse interlinked types of damage at various scales. Ice crystals growing extracellularly create cell dehydration through the reduction of available liquid water³. When exposed to prolonged freezing temperatures, the formation of larger ice masses through the recrystallization process can mechanically affect plant tissues and internal structures⁴. Facing these challenges, we tried to find the best strategy to preserve the most vulnerable organs and tissues by producing specific protectants at the plant surface.

Ice-Binding Proteins (IBPs) have the ability to bind ice crystals and operate different actions on them. Among this particular protein family, Antifreeze Proteins (AFPs) encircle crystals to inhibit their growth. Ice-Nucleation Proteins (INPs), on the other hand, act as nucleation cores to promote ice formation⁵. These apparently contradictory actions rely on similar mechanisms help reducing frost damage in their own way.

Our first strategy is the most intuitive: producing antifreeze proteins at the plant surface to inhibit crystal growth. Part of natural protection mechanisms in wintering plants, these proteins bind ice crystals and limit their expansion ⁶. Since external and internal ice crystal formation seem to be interrelated⁷, the prevention of ice crystal formation externally could have a real impact on inhibiting recrystallisation, and lowering the water freezing temperature.

Among the numerous AFP types expressed in Nature, a particular AFP type called RiAFP, taken from the freezing-avoiding beetle Rhagium inquisitor, seems particularly suitable for our objective in terms of action and synthesis requirements. When expressed at 15°C, its high thermal hysteresis activity could significantly reduce the risk of ice formation and indirectly protect inner tissues ⁸.

What is also interesting with AFPs is the perspectives offered by protein engineering and isoform synergistic effect. The latter refers to the fact that different AFP isoforms operating together give a better antifreeze efficiency than the sum of the same isoforms taken separately. These two strategies enhance the protein activity, making these compounds even more interesting ⁹.

Our second strategy is opposed to the first one, but could be of equally interest. Producing ice-nucleating proteins at the plant surface to favor ice formation. This is the basic principle of water aspersion, one of the most commonly techniques used by wine-growers to protect vines from frost. The mechanism relies on the release of latent heat from this exothermic reaction as well as the insulating properties of the ice layer formed ¹⁰. Coupled to the aspersion method, ice-nucleating proteins could significantly optimize the process and allow high water savings.

We are not the first ones to consider modulating ice crystal formation to protect plants in the context of the IGEM competition. As an example, the project “PlantiFreeze” of 2014 Weston team consists of the production and secretion of AFP by E.coli. (https://2014hs.igem.org/Team:CSWProteens)

Additionally, in the 1980’s, a product called Frostban was developed by using genetically modified ice-nucleating bacteria. The goal was to knock-out the Ice-nucleation protein gene to engineer a replacement for naturally occurring ice-nucleating bacteria that were on the plant leaves and were responsible for frost damage. The strategy was different from ours because it relied on massive spraying of GMOs that were supposed to compete with their natural counterpart¹¹. While the product was proven to be efficient, numerous GMO related controversies made it impossible to commercialise. We did a case-study of Frostban in our Wiki here, do not hesitate to go there if you want more details!

Heat Protection

At high temperatures around 40°C, many crops including grapevines suffer from protein denaturation, as well as water stress and solar radiation damage. Despite automatic stomatal closure, water loss by evapotranspiration can cause severe dehydration of tissues ¹². In addition to temperature increases, a direct exposure to solar radiation enhances the number of harmful free radicals inside the cells and can damage them. Shriveled fruits are a main consequence of solar damage ¹³. Protecting grapevines from excess sunlight could significantly help farmers to deal with climate change, leading to an increase in the frequency of heatwaves and droughts.

Some plants and animals have the property to adapt their albedo, the ability of a surface to reflect sunlight, according to their environment. The desert plant Encelia farinosa produces leaves covered with white trichomes during the drought season. This induces a considerable albedo increase and reduces the heat loading of leaves, limiting water loss and high temperature stress ¹².

We hypothesized we may reproduce this albedo augmentation by creating a physical barrier against solar radiations. This can be achieved by making our microorganism produce white reflective compounds at the leaf surface. The challenge was to find white molecules that could be expressed on the plant without compromising its functions or the existing microbiota.

We tried to find our inspiration in nature, and it turned out that different compound types take a white coloration. Proteins such as casein, main protein of milk, are already used for industrial purposes and could be of interest for this objective¹⁴. However, their nutritive nature could potentially disturb the regular biodiversity¹⁵. Biopolymers such as chitin and its derivatives also own interesting reflecting properties, and appear neutral for the plant ¹⁶. Another interesting approach is the biomineralization: some bacteria can precipitate mineral ions like calcium carbonate and cover surfaces with a white layer ¹⁷.

Fighting against heat-related crop damages through albedo modification is an area of active research. For example, a product called Invelop composed of talc was developed to reduce exposure to solar radiation and also to fight against certain parasites¹⁸. Reflective surfaces are even engineered to cool down rooftops! ¹⁹

Want to learn more about the effects of extreme temperatures on plants, and our different research approaches to protect them? Take a look to our complete written report where the choice of the compounds is discussed (click on the following image!):

References

Martinson T & Martin G, How Grapevines Reconnect in the Spring, Grapes 101, 2012.
Frioni T. et al, Impact of spring freeze on yield, vine performance and fruit quality of Vitis interspecific hybrid Marquette, Scientia Horticulturae Volume 219, pp. 302-309, 2017.
Report from Food and Agriculture Organization, Frost Protection: fundamentals, practice and economics Vol. 1, Snyder RL & de Melo-Abreu JP, 2004.
Pearce RS, Plant Freezing and Damage, Annals of Botany 87: 417-424, 2001.
Bar Dolev M et al, Ice-Binding Proteins and Their Function, Annual Review of Biochemistry, 2016.
Gupta R & Deswal R, Antifreeze proteins enable plants to survive in freezing conditions, Journal of Biosciences;39(5):931-44, 2014.
Wisniewski M et al, Observations of Ice Nucleation and Propagation in Plants Using Infrared Video Thermography, Plant Physiology 113: 327-334, 1997.
Report from Food and Agriculture Organization, Frost Protection: fundamentals, practice and economics Vol. 1, Snyder RL & de Melo-Abreu JP, 2004.
Hopkins, W. and Hüner, N. (2014). Introduction to plant physiology. Hoboken (NJ): John Wiley & Sons
Krasnow MN et al, Distinctive symptoms differentiate four common types of berry shrivel disorder in grape, California Agriculture, 64(3), 2010.
>de Kruif C. et al, Casein micelles and their internal structure, Advances in Colloid and Interface Science 171-172, pp.36-52, 2012.
Wang Y. et al, Growth of marine fungi on polymeric substrates, BMC Biotechnology, 2016.
Periasamy Anbu, Chang‑Ho Kang, Yu‑Jin Shin and Jae‑Seong So, Anbu et al. SpringerPlus, Formations of calcium carbonate minerals by bacteria and its multiple applications, 2016.
Adrienne J. Phillips, Robin Gerlach, Ellen Lauchnor, Andrew C. Mitchell, Alfred B. Cunningham & Lee Spangler, Engineered applications of ureolytic biomineralization: a review, Biofouling: The Journal of Bioadhesion and Biofilm Research, 2014.

Igem ionis

Is an association created by Sup’Biotech student in 2015. Since this first participation, two teams (2015 and 2016) won the gold medal and several nominations: « Best presentation », « Best applied design », and « Best environmental project ».
The strength of the IGEM IONIS comes from its multidisciplinarity and its complementarity.

This year we are 20 members from different schools:
18 students from Sup’Biotech
1 student from e-artsup
1 student from Epita
Read more …