In the wilds of the laboratory, Camilo Villouta captures the fleeting effects of freezing water where flowers form.

When studying how plants in winter deal with freezing temperatures, the observation of ice formation in their tissues is especially elusive. I addressed this topic in my dissertation, which challenged me to find new methods to answer them. At the time, I was a PhD student at the fruit crops lab at the University of Wisconsin-Madison, under the guidance of Dr. Amaya Atucha. Our goal was to understand the development of cold hardiness in the terminal buds of cranberry (Vaccinium macrocarpon). Most importantly, we wanted to know what strategy they used to withstand and survive ice formation.

In woody perennial flowering species, buds are known to survive exposure to freezing temperatures by either of two freezing survival strategies. Both follow a similar principle: avoid the formation of ice crystals with their potential of causing lethal damage in the flower primordia, tissues that are the precursor of the mature flower. These strategies have extravagant names, which nonetheless describe their main characteristics very well. On one hand, we have “deep supercooling,” where buds avoid the propagation of ice into the flower primordia, keeping them ice-free even when they are at subfreezing temperatures. The other strategy is named “extraorgan freezing,” where the flower primordia dehydrate, pushing water away from themselves towards the ice-tolerant bud scales. In the case of cranberry terminal buds, we needed to determine which strategy was at work, both to interpret damage patterns more accurately and to know which techniques to use for assessing cold-hardiness levels at different times across the season.

While designing the study to answer this question, we reviewed a range of current methods for imaging plant tissues, assessing them for their ability to capture freezing events inside an intact bud. Thermal video recording offers only a surface view of freezing progression, as do electric thermometers, or thermocouples, which only register temperature changes at a specific location. MicroCT (computed tomography) scans offer great resolution of internal tissues, but cannot track freezing, as the method does not detect a contrast difference between liquid water and ice. Magnetic resonance imaging (MRI), however, can detect this difference. Searching the UW campus for appropriately-sized MRI machines, I found one in use at the Small Animal Imaging Center. While a regular MRI has an entrance designed to fit a person, this apparatus was made for animals such as rats, with an entrance diameter of about 3 inches.

Once we found the proper MRI machine, we needed to find a way to control the rate of decreasing temperature at which the buds would have been exposed—a rate of vital importance. Studies have reported that a common rate of temperature change in nature is 1 °C/hr (degrees Celsius per hour). Thus in laboratory conditions, we usually work with rates ranging from 1 to 4 °C/hr, otherwise you risk creating artificial effects in non-realistic freezing conditions. To detect the progress of freezing in our plants, I needed a device to control temperature with great precision, which I set out to construct. The goal was to acquire MRI images corresponding to slices of our samples at room temperature, and then slowly decrease the temperature, capturing other images at just below freezing, and then two more times at colder temperatures, always of the same slices. In collaboration with the Morgridge Institute’s Advanced Fabrication Laboratory,— a.k.a. the “Fab Lab”—at UW Madison, we started to develop a prototype. It needed to follow several guidelines: it could not have any of its metallic implements close to the MRI apparatus, while at the same time it had to withstand freezing temperatures, exhibit chemical resistance, and not interact with the imaging process. The prototype needed to have three compartments: one for the flow of chilled glycol, a second for the addition of a chemical that helps with image contrast, and a third to surround the samples with all these compounds. Once the design was defined, we started testing the circulating system. We located the circulating bath and pump for the chilled glycol with their metallic parts outside the room, connecting the glycol via tubing with the prototype inside the MRI. With this circulation system exposed to air, frost formation was also a big concern, as this could damage the expensive MRI equipment. We ran trials in our lab just to see where frost was forming and find corrective measures.

I needed a device to control temperature with great precision, which I set out to construct.

By this time, a year had passed, and finally, we were able to obtain our first images. After doing the official runs in late fall of 2019, I measured the signal intensity at different regions of the buds and compared them across the different imaged temperatures. Higher signal intensity translated into a higher presence of liquid water, and the opposite meant relocation or freezing of water. My analysis showed a gradual decrease in the amount of liquid water in the internal tissues of the bud. This was an important piece of evidence for our study. With the MRI, we observed a range of freezing events occurring at temperatures below -20 °C, while in visual damage evaluations for that same date, we found that damage did not start until reaching temperatures of -24 °C and lower.

In the end, our study concluded that terminal buds of cranberry survive exposure to freezing temperatures by undergoing a process of freeze dehydration, a variant of the extraorgan freezing survival strategy. From here, we can direct our efforts on developing cold hardiness models with a greater understanding of the damage patterns, knowing what mechanisms need to be developed during the fall during the seasonal acclimation to winter. This knowledge will help farmers and horticulturists decide how to respond when fruit crops are exposed to the threat of unseasonably cold temperatures—conditions likely to occur with greater frequency as climate change advances.

Camilo Villouta is a Putnam Postdoctoral Fellow at the Arnold Arboretum.