The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Summary

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Abstract

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

Introduction

Freezing temperatures that occur when plants are actively growing can be lethal, particularly if the plant has little or no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic and arctic ecosystems ^1-6. Episodes of severe spring frosts have had major impacts on fruit production in the USA and South America in recent years ^7-9 and have been exacerbated by the early onset of warm weather followed by more typical mean low temperatures. The early warm weather induces buds to ....

Protocol

1. Preparation of Plant Materials

1. Use either leaves or whole plants of subject plant material (Hosta spp. or Phaseolus vulgaris).

2. Preparation of Water Solutions Containing Ice Nucleation Active (INA) Bacteria

1. Culture the INA bacterium, Pseudomonas syringae (Strain Cit-7) in petri dishes at 25 °C on Pseudomonas Agar F prepared with 10 g/L of 100% glycerol per the manufacturer’s direction.
2. After cultures have grown sufficiently, place at 4 °C until needed but keep at 4 °C for two days prior to ensure a high level of ice nucleation activity.
3. S....

Results

Ice-nucleating activity of the Ice+ bacterium, Pseudomonas syringae (strain Cit-7)

A 10 µl drop of water and 10 µl of water containing P. syringae (Cit-7) were placed on the abaxial surface of a Hosta leaf (Hosta spp.) (Figure 4). As illustrated, the drop of water containing the INA bacteria froze first and was responsible for inducing the leaf to freeze while the drop of water on the leaf surface remained unfrozen.

Freezi.......

Discussion

Water has the ability to supercool to temperatures well below 0 °C and the temperature at which water will freeze can be quite variable.³⁶ The temperature limit for supercooling of pure water is about -40 °C and is defined as the homogeneous nucleation point. When water freezes at temperatures warmer than -40 °C it is brought about by the presence of heterogenous nucleators that enable small ice embryos to form which then serve as a catalyst for ice formation and growth.³⁷ There are a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Infrared Camera	FLIR	SC-660	Many models available depending on application
Infrared Analytical Software	FLIR	ResearchIR 4.10.2.5	$3,500
Pseudomonas syringae (strain Cit-7)			Kindly provided by Dr. Steven Lindow, University of California  Berkeley icelab@berkeley.edu
Pseudomonas Agar F	Fisher Scientific	DF0448-17-1