This research was funded by the Austrian Science Fund (FWF): P23681-B16.

1. Preparation of Plant Materials
<ol>
	<li>Use either leaves or whole plants of subject plant material (Hosta spp. or Phaseolus vulgaris).</li>
</ol>
2. Preparation of Water Solutions Containing Ice Nucleation Active (INA) Bacteria
<ol>
	<li>Culture the INA bacterium, Pseudomonas syringae (Strain Cit-7) in petri dishes at 25 &#176;C on Pseudomonas Agar F prepared with 10 g/L of 100% glycerol per the manufacturer&#8217;s direction.</li>
	<li>After cultures have grown...

<ol>
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Res. 40 (4), 660-670 (2008).</li><li>Hacker, J., Spindelb&#246;ck, J., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesophyll+freezing+and+effects+of+freeze+dehydration+visualized+by+simultaneous+measurement+of+IDTA+and+differential+imaging+chlorophyll+fluorescence.">Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence.</a> Plant Cell Environ. 31, 1725-1733 (2008).</li><li>Neuner, G., XU, B., Hacker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Velocity+and+pattern+of+ice+propagation+and+deep+supercooling+in+woody+stems+of+Castanea+sativa.,+Morus+nigra.+and+Quercus+robur.+measured+by+IDTA.">Velocity and pattern of ice propagation and deep supercooling in woody stems of Castanea sativa., Morus nigra. and Quercus robur. measured by IDTA.</a> Tree Physiol. 30, 1037-1045 (2010).</li><li>Hacker, J., Ladinig, U., Wagner, J., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflorescences+of+alpine+cushion+plants+freeze+autonomously+and+may+survive+subzero+temperatures+by+supercooling.">Inflorescences of alpine cushion plants freeze autonomously and may survive subzero temperatures by supercooling.</a> Plant Sci. 180, 149-156 (2011).</li><li>Kuprian, E., Briceno, V., Wagner, J., Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+barriers+promote+supercooling+and+prevent+frost+injury+in+reproductive+buds,+flowers+and+fruits+of+alpine+dwarf+shrubs+throughout+the+summer.">Ice barriers promote supercooling and prevent frost injury in reproductive buds, flowers and fruits of alpine dwarf shrubs throughout the summer.</a> Env. Exp. Bot. 106, 4-12 (2014).</li><li>Wisniewski, M., Glenn, D. M., Fuller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+hydrophobic+particle+film+as+a+barrier+to+extrinsic+ice+nucleation+in+tomato+plants.">Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants.</a> HortScience. 127, 358-364 (2002).</li><li>Aryal, B., &amp;Neuner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+wettability+decreases+along+an+extreme+altitudinal+gradient.">Leaf wettability decreases along an extreme altitudinal gradient.</a> Oecologia. 162, 1-9 (2010).</li><li>Wisniewski, M., Fuller, M. P., Margesin, R., Schinner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ice nucleation and deep supercooling in plants: new insights using infrared thermography. In: Cold adapted organisms. Ecology, physiology, enzymologyandmolecularbiology. , 105-118 (1999).</li><li>Franks, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biophysics and biochemistry at low temperatures. , (1985).</li><li>Neuner, G., Kuprian, E., Hincha, D., Zuther, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+thermal+analysis+of+plant+freezing+processes.">Infrared thermal analysis of plant freezing processes.</a> Methods in Molecular Biology: Plant Cold Acclimation. , 91-98 (2014).</li></ol>

Water has the ability to supercool to temperatures well below 0 &#176;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 &#176;C and is defined as the homogeneous nucleation point. When water freezes at temperatures warmer than -40 &#176;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...

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 break, activating the growth of new shoots, leaves, and flowers all of which have very little to no frost tolerance 1,3,10-12. Such erratic weather patterns have been reported to be a direct reflection of ongoing climate change and are expected to be a common weather pattern for the foreseeable future 13. Efforts to provide economical, effective, and environmentally-friendly management techniques or agrochemicals that can provide increased frost tolerance have had limited success for a host of reasons but this can be partly attributed to the complex nature of freezing tolerance and freezing avoidance mechanisms in plants. 14
The adaptive mechanisms associated with frost survival in plants have traditionally been divided into two categories, freezing tolerance and freezing avoidance. The former category is associated with biochemical mechanisms regulated by a specific set of genes that allow plants to tolerate the stresses associated with the presence and the dehydrative effect of ice in its tissues. While the latter category is typically, but not solely, associated with structural aspects of a plant that determine if, when, and where ice forms in a plant 14. Despite the prevalence of freeze avoidance as an adaptive mechanism, little research has been devoted in recent times to understanding the underlying mechanisms and regulation of freeze avoidance. The reader is referred to a recent review 15 for greater details on this subject.
While the formation of ice at low temperatures may seem like a simple process, many factors contribute to determining the temperature at which ice nucleates in plant tissues and how it spreads within the plant. Parameters such as the presence of extrinsic and intrinsic ice nucleators, heterogeneous vs. homogeneous nucleation events, thermal-hysteresis (antifreeze) proteins, the presence of specific sugars and other osmolytes, and a host of structural aspects of the plant can all play a significant role in the freezing process in plants. Collectively, these parameters influence the temperature at which a plant freezes, where ice is initiated and how it grows. They can also affect the morphology of the resulting ice crystals. Various methods have been used to study the freezing process in plants under laboratory conditions, including nuclear magnetic resonance spectroscopy (NMR) 16, magnetic resonance imaging (MRI) 17, cryo-microscopy 18-19, and low-temperature scanning electron microscopy (LTSEM). 20 Freezing of whole plants in laboratory and field settings, however, has mainly been monitored with thermocouples. The use of thermocouples to study freezing is based on the liberation of heat (enthalpy of fusion) when water undergoes a phase transition from a liquid to a solid. Freezing is then recorded as an exothermic event. 21-23 Even though thermocouples are the typical method of choice in studying freezing in plants, their use has many limitations that limit the amount of information obtained during a freezing event. For instance, with thermocouples it is difficult to nearly impossible to determine where ice is initiated in plants, how it propagates, if it propagates at an even rate, and if some tissues remain free of ice.
Advances in high-resolution infrared thermography (HRIT) 24-27, however, have significantly increased the ability to obtain information about the freezing process in whole plants, especially when used in a differential imaging mode. 28-33 In the present report, we describe the use of this technology to study various aspects of the freezing process and various parameters that affect where and and at what temperature ice is initiated in plants. A protocol will be presented that will demonstrate the ability of the ice-nucleation-active (INA) bacterium, Pseudomonas syringae (Cit-7) to act as an extrinsic nucleator initiating freezing in a herbaceous plant at a high, subzero temperature.
High-resolution Infrared Camera
The protocol and examples documented in this report utilize a high resolution infrared video radiometer. The radiometer (Figure 1) supplies a combination of infrared and visible spectrum images and temperature data. The spectral response of the camera is in the range of 7.5 to 13.5 &#181;m and provides 640 x 480 pixel resolution. Visible spectrum images generated by the built-in camera can be fused with IR-images in real time, which facilitates the interpretation of complex, thermal images. A range of lenses for the camera can be used to make close-up and microscopic observations. The camera can be used in a stand-alone mode, or interfaced and controlled with a laptop using propietary software. The software can be used to obtain a variety of thermal data embedded in the recorded videos. It is important to note that a wide variety of infrared radiometers are commercially available. Therefore, it is essential that the researcher discuss their intended application with a knowledgeable product engineer and that the researcher test the ability of any specific radiometer to provide the information needed. The imaging radiometer used in the described protocol is placed in an acrylic box (Figure 2) insulated with Styrofoam in order to deter exposure to condensation during the warming and cooling protocols. This protection is not needed for all cameras or applications.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Infrared Camera</td>
			<td>FLIR</td>
			<td>SC-660</td>
			<td>Many models available depending on application</td>
		</tr>
		<tr>
			<td>Infrared Analytical Software</td>
			<td>FLIR</td>
			<td>ResearchIR 4.10.2.5</td>
			<td align="right">$3,500</td>
		</tr>
		<tr>
			<td rowspan="2">Pseudomonas syringae (strain Cit-7)</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2"><a href="mailto:icelab@berkeley.edu">Kindly provided by Dr. Steven Lindow, University of California&#160; Berkeley icelab@berkeley.edu</a></td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Pseudomonas Agar F</td>
			<td>Fisher Scientific</td>
			<td>DF0448-17-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of high-resolution infrared thermography (hrit) for the study of ice nucleation and ice propagation in plants

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.

Ice-nucleating activity of the Ice+ bacterium, Pseudomonas syringae (strain Cit-7)
A 10 &#181;l drop of water and 10 &#181;l 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.
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The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

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).

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

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 ...

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9.5K Views.  Agricultural Research Service (USDA-ARS), Kearneysville, WV. 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).