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 sufficiently, place at 4 &#176;C until needed but keep at 4 &#176;C for two days prior to ensure a high level of ice nucleation activity.</li>
	<li>Scrape bacteria from a single plate from the surface of the agar with a plastic, disposable or re-usable metal spatula at the time of use and place in 10-15 ml of deionized water in a 25 ml disposable cuvette. The concentration should be in the range of 1 x 107 to 1 x 109 &#183; ml -1. The solution will appear cloudy. There is no need to confirm the concentration using a hemocytometer or spectrophotometer, as concentration need only be approximate.</li>
	<li>Vortex the cuvette for a minimum of 10 sec to distribute the bacteria. 
	Note: The specific concentration of the resulting INA mixture is not important and the protocol described will provide more than an adequate level of ice nucleation activity. This mixture of INA bacteria and water will be used later in the nucleation experiments.</li>
</ol>
3. Setting Up a Freezing Experiment
<ol>
	<li>Place the high resolution infrared camera (SC-660) inside the protective acrylic box so that the lens projects through the opening in the front of the box and the wires connecting the camera to a laptop or recording device exit through the rear opening of the box. Secure the lid of the box and place the box inside the environmental chamber or freezer in a location that will allow the subject plant material to be seen.
	<ol>
		<li>Provide a dark background around the plant material by lining the walls of the chamber with black construction paper to prevent interference from reflected infrared energy.</li>
		<li>Fit the chamber with LED lighting to minimize heating from the light source when recording images in visible wavelengths is required. Only a minimum of lighting, such as a battery-operated closet light or other small LED device, is required for the plants to be visible by the camera.
		<ol>
			<li>Once visible images of the subject plant material are taken, turn off the LED lighting. Distribute all external wired connections (firewire connection to computer, power cord, etc.) to the camera via a port or other opening in the chamber.</li>
		</ol>
		</li>
		<li>Fill any extra space in the port or opening with insulating foam material to avoid or reduce temperature gradients within the chamber. Set the initial temperature of the chamber at 1 &#176;C.</li>
	</ol>
	</li>
	<li>Align plants or plant parts so that the plant material is in the field-of-view of the camera and the plant material is visible on the remote viewing screen or within the chosen software.</li>
	<li>Allow plants to equilibrate at 1 &#176;C for 30 min to 1 hr, depending on the size of the plant material, prior to initiating a controlled freezing experiment. This ensures that the temperature of the plant will not lag behind air temperature by many degrees once the freezing experiment is initiated. Equilibration is achieved when the temperature of the plant material is within 0.5 &#176;C of air temperature.
	<ol>
		<li>Place a layer of Styrofoam insulation on top of the soil of potted plants if potted plants are used. Once the plants have equilibrated, commence cooling of the chamber. 
		Note: The layer of insulation on the soil surface of the pot reduces the amount of continued heat loss from the pot to the air surrounding the plant, and prevents the roots from freezing, as this would not typically occur during a frost event in nature due to the massive reservoir of residual heat present in the soil.</li>
	</ol>
	</li>
	<li>Set the desired camera parameters (color palette, temperature range, specific areas of interest, etc.), as discussed in 3.4.1-3.4.4.
	<ol>
		<li>Select the rainbow palette to display the temperature variations while viewing the live image.</li>
		<li>Set the temperature span to 5 &#176;C by adjusting the temperature bar located just below the image in the software.</li>
		<li>Choose the linear scale (algorithm) for converting the infrared data into the false color image as defined by the selected palette (rainbow) and set the range of temperature to 5 &#176;C and to track automatically based on the image. Alternatively, adjust the set range manually while conducting the experiment.
		<ol>
			<li>Use the temperature of a specific point or an average temperature within the defined area of interest provided by the software. Retrieve the temperature data of all pixels from the recorded video sequence or from the information embedded in the image file. Figure 3 shows a typical screenshot from within ResearchIR software.</li>
		</ol>
		</li>
		<li>Place a cursor on a location on the plant tissue that represents a specific point of interest. Define the area of interest as points (1 -3 pixels in size), boxes, lines, ellipses, or circles. Multiple combinations of points or shapes can be located over the image.</li>
	</ol>
	</li>
	<li>Recording a video sequence
	<ol>
		<li>Set the camera to record at 60 Hz and for the recording to be stopped manually.</li>
		<li>Indicate the location on the computer or external drive where the recorded video file will be placed.</li>
		<li>Commence recording. 
		Note: Recording to an external hard drive is highly recommended since large video files will be generated. Recorded video files can be later edited to contain only the portion containing the necessary information. This will greatly reduce the file size.</li>
		<li>Lower the temperature of the chamber incrementally by 0.5 -1.0 &#176;C. Wait until the plant temperature equilibrates with air temperature and then lower the temperature again by 0.5-1.0 &#176;C. Depending on the mass of the plant tissue being observed and its morphology, equilibration can take 10 to 15 min. Thus, giving a cooling rate of about 4 &#176;C/hr.</li>
		<li>Continue in this manner until the plant freezes and observations are completed. End the recording when the freezing process has been completed. 
		Note: The plant tissue has equilibrated with air temperature when the plant material and background are the same color since they are at the same temperature. Since the background temperature and the temperature of the plant tissue are the same, it may be difficult to visualize the plant material until you again lower the temperature and there is temperature differential between the plant tissue and air temperature.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests or conflicts of interest.

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 multitude of molecules in nature that act as very efficient ice nucleating agents, thus most freezing of water in nature occurs at temperatures just below 0 &#176;C. The ability to regulate or influence the activity of heterogenous nucleating agents has significant potential as a novel approach to providing frost protection to plants. Understanding how ice forms and propagates in freezing sensitive and freezing-tolerant plants is essential to achieving this objective.
As indicated in the Introduction, various methods have been used to study the freezing process in plants under laboratory conditions, however, freezing of plants in nature has mainly been monitored with the use of thermocouples. High-resolution infrared thermography (HRIT) 24-28,34, offers several distinct advantages as a method for studying the freezing process in plants. HRIT allows one to observe the initial site of ice formation, the number of freezing events needed to freeze an entire plant, actually observe how ice is propagated in a plant and if any barriers to ice propagation are present, and determine if any portions of a plant remain ice free. Most importantly, it allows one to observe the freezing process in whole plants rather than small, isolated portions of a plant that have been removed from the parent plant.
The present report outlines the application of HRIT to the study of freezing in intact plants or plant parts, and provides several examples of how this technology can be used to examine several parameters that can influence how and when ice forms in plants, and how ice is propagated. Critical aspects of conducting these studies involve the sensitivity and accuracy of the infrared camera, the parameters used in the setup of the camera and the recording of video sequences, the cooling rate, the structural/morphological complexity of the subject being viewed, and knowledge about infrared science. These items will be addressed individually.
Sensitivity and accuracy of the infrared camera (radiometer)
The exothermic events during freezing of plant tissues that are being visualized are very small, ranging from &#60;0.1 to about 0.5 &#176;C. Therefore the infrared camera must be sensitive enough to easily differentiate small changes in temperature. Temperature accuracy is also an important aspect and requires that the camera is calibrated on a regular basis (at least once a year). While this can be done by the user, it requires the use of several black bodies covering a wide range of temperatures. Therefore, it is best to have the camera factory-calibrated. If a high level of temperature accuracy is absolutely essential, it is highly recommended that a thermocouple be used in conjuction with the infrared camera. This can be mounted near the object being studied to give an accurate estimate of air temperature.
Camera parameters
A host of parameters can be adjusted on advanced, high-end infrared cameras. In using the camera to view and/or record freezing events, it is important that image averaging be used in order to reduce a noisy image, thus making it easier to visualize plant parts and freezing events. Image averaging occurs when a high quality image is selected in the camera settings. Since minor freezing exotherms are expected, it is also important when viewing the freezing process to set the temperture span of the camera to cover a small temperature range (2-5 &#176;C). This is needed because the software will distribute the selected color pallette over the full span set for the camera. Therefore, if there are 10 colors in the pallette and one has the span set to 100 &#176;C, their would only be a change in color if there was a 10 &#176;C change in temperature. A high capture rate (ten frames per second) should be used so that small exothermic events, which dissipate quickly, are not missed. Different color palettes and grey scales can be selected from a drop down menu. Selection of the most appropriate palette should be based on whether or not it provides the best option for visualizing the thermal events of interest. Advanced cameras also offer several options for recording a video sequence and/or capturing single images. A specific number of frames over a set time duration can be selected. This is best for recording sequences of short duration (minutes) rather than hours. Alternatively, the number of frames per second can be indicated and the camera set to stop recording manually or after a specific number of frames. Advanced cameras also offer the option for recordings to commence or end based on pre-defined triggers (temperature or time).
Cooling rate
It is important that the temperature of plant material being viewed does not differ dramatically from the air temperature during cooling. If the temperature is lowered too quickly, plants will supercool and freeze at a lower air temperature than they would under natural cooling rates. Most studies recommend a cooling rate of 1-2 &#176;C hr-1, especially at temperatures above -5 &#176;C, which provides ample time for plants to come to equilibrium with the air temperature. In actuality, the plant material may come into equilibrium much faster. This can be determined by comparing the temperature of the plant material with the temperature of the background around the plant. If the plant is in equilibrium, it will be difficult to discern the plant from its background in the infrared image as it will be at the same temperature as the background and the image will appear to be nearly homogeneous in color.
Structural\morphological complexity of the object being viewed
Since the images being viewed represent images of temperature, objects that overlap will appear as contiguous objects rather than discrete objects. This can make discerning where freezing events are occuring very difficult and also increase the difficulty in determining how ice is being propagated in the plant. The best way to deal with this problem is to first work with simple objects (individual leaves, stems, etc.) and then build up to more complex objects. Experience in working with specific material has great value in dealing with this problem. Additionally, the ability to overlay the infrared image on top of a digital, visible-light image can also greatly assist in analyzing and understanding the infrared data.
Knowledge of infrared science
Although it would be advantageous to be able to simply point the camera at an object and know that the temperature data received is 100% accurate, understanding how infrared energy interacts with its environment can greatly increase ones understanding of how best to use the research-grade infrared cameras and interpret the data. One should become somewhat familiar with the terms emissivity, reflectance, and absorbance. For the most part, the camera can be used without worrying about these parameters, however, they can help to explain the nature of the image being displayed and its overall quality and accuracy. Briefly, when infrared energy hits an object it can be either reflected or absorbed and then emitted. The nature of the object being studied, therefore, can affect the accuracy of the data being received. If an object has a high reflectance, one will receive an image more representative of the surrounding objects that are emitting infrared energy than the object itself. Absorbance of infrared energy without emitting the infrared energy can also lead to obtaining false temperature data from the object being studied. The camera sensors detect emitted infrared energy, therefore, the most accurate temperatures are obtained from objects that have a high level of emissivity. Fortunately, plants do have a high level of emissivity allowing accurate temperature measurements. Lower levels of emissivity can be compensated for by adjusting this parameter in the camera settings which will then use an algorithm to make an appropriate adjustment in the temperature readout.
The ability to accurately determine how and when plants freeze is essential to understanding the evolution of freeze-avoidance mechanism and the role of plant structure in the freezing process. Freezing, despite its apparent simplicity, is a complex process and plants have evolved a host of structural adaptations to avoid freezing, compartmentalize ice formation, and prevent the propagation of ice. High resolution infrared thermography is a novel and powerful tool that can be used to study the complexity of the freezing process in plants and lead to the development of effective new methods of frost protection. A better understanding of freeze-avoidance can also help us to understand how these adaptive mechanisms have evolved, and the role they play in the biology and survival of different plant species.

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.
Freezing and Ice Propagation in a Woody Plant
Figure 5 illustrates both ice initiation and ice propagation in a stem of oak (Quercus robur). Ice formation was initiated in the vascular cambium\phloem region of the stem and propagated circumferentially around the stem. The rate of ice propagation in woody plant stems is much greater in a longitudinal direction than in a lateral and circumferential direction.31
Rates of Ice Propagation and Barriers to Ice Propagation
Ice was initiated in the stem of a bean plant (P. vulgaris) at the site where INA bacteria had been placed (Figure 6A, arrow). Following the initial freezing event, ice propagated up and down the stem (Figure 6B-C). Using the video sequence, which has a time stamp, and measuring distance on the stem, enables one to calculate the rate of ice propagation over a given distance. The graph in Figure 6 presents the rate of ice propagation up the stem of the bean plant from the point of initial freezing and illustrates a decreased rate of ice propagation as ice passes through the nodal region of the plant. Using infrared thermography also allows one to determine the presence of any physical barriers that prevent the propagation of ice into specific tissues. Figure 7 illustrates freezing in an alpine species, Loiseleuria procumbens, where the vegetative part (stem and leaves) of the plant has frozen but the terminal flower buds remain unfrozen. Ice formation did not occur until 126-164 min after freezing of the stem and leaves had occurred and the resulting exothermic response had dissipated. As reproductive shoots of alpine woody species are freezing sensitive 3,33, freezing avoidance is crucially important to reproductive success.
Ability of Hydrophobic Barriers to Block Extrinsic Ice Nucleation Induced Freezing
A tomato plant (Solanum lycopersicum) was coated with a hydrophobic kaolin-based material (Figure 8A) in order to determine if the hydrophobic barrier could block extrinsic ice nucleation induced freezing. The degree of contact of droplets of liquid with the leaf surface was much greater in uncoated leaves (Figure 8B) compared to coated leaves (Figure 8C). As illustrated in Figure 8D, uncoated plants (right) exhibited an exothermic event typical of a freezing event, while coated plants (left) remained unfrozen and supercooled to approximately -6.0 &#176;C. Details of these experiments can be found in Wisniewski et al. 34 A trend of greater hydrophobicity in the leaf structure of native plant species along an altitudinal gradient has been noted by Aryal and Neuner. 35
<img alt="Figure 1" src="/files/ftp_upload/52703/52703fig1.jpg" /> 
Figure 1.&#160;High-resolution Infrared Radiometer. The model illustrated is a FLIR SC-660 Infrared Video Camera.
<img alt="Figure 2" src="/files/ftp_upload/52703/52703fig2.jpg" /> 
Figure 2.&#160;Protective enclosure for the infrared camera. Anacrylic box is used to house the camera and prevent condensation from forming on the infrared camera during freezing and thawing experiments. (A) Box with top removed. (B) Camera inserted in the acrylic box and lid closed.
<img alt="Figure 3" src="/files/ftp_upload/52703/52703fig3.jpg" /> 
Figure 3.&#160;Viewing and Analyzing Infrared Images and Remote Camera Control. Screen shot from the ResearchIR software. The software is used to view the live image, change camera settings, record single images, make video recordings, and analyze temperature data in the images. Insert on right shows options for changing camera settings while insert on lower left shows a temperature histogram of the live image.
<img alt="Figure 4" src="/files/ftp_upload/52703/52703fig4.jpg" /> 
Figure 4.&#160;Extrinsic nucleation induced freezing of Hosta leaf (Hosta spp.). Unfrozen droplets of water and INA bacteria, Pseudomonas syringae (strain Cit-7), are present on the abaxial surface of the leaf (A). INA droplet freezes first (B) and initiates freezing of the leaf (C). Ice spreads throughout the leaf (D) and despite the freezing of the leaf, the water droplet remains unfrozen (E). Droplet of water on surface of leaf freezes after the entire leaf has frozen and is beginning to cool at its edges (F).
<img alt="Figure 5" src="/files/ftp_upload/52703/52703fig5.jpg" /> 
Figure 5.&#160;Ice initiation and propagation in the stem of a woody plant (Quercus robur). Left Panel: Cross-section of a woody stem of oak. (A-H) Initiation of freezing event in the region of the phloem and vascular cambium (A) and progression of ice formation around the stem (B&#8211;H). Kuprian and Neuner, unpublished.
<img alt="Figure 6" src="/files/ftp_upload/52703/52703fig6.jpg" /> 
Figure 6.&#160;Rate of ice propagation in a bean plant (Phaseolus vulgaris) calculated using high-resolution infrared thermography. (A) Ice initiated in stem (arrow). (B&#8211;C) Ice propagation up and down the stem. Graph at top of figure displays the rate of ice propagation presented as the distance ice traveled over time as it moved up the stem from the original site of freezing. A delay in ice propagation occurred as ice moved through the nodal portion of the plant stem. This figure was modified from Wisniewski et al.24
<img alt="Figure 7" src="/files/ftp_upload/52703/52703fig7.jpg" /> 
Figure 7.&#160;Barrier to ice formation in the alpine woody plant, Loiseleuria procumbens (alpine azalea). (A) Visible light image of the stem of alpine azalea showing central stem, attached leaves, and terminal buds. (B&#8211;C) Freezing is initiated in the stem and ice propagates out into leaves. Terminal buds remain unfrozen. (D&#8211;E) Terminal buds freeze independently 126-164 min after initial freezing of stem and leaves. During this time the heat of enthalpy produced by the freezing of the stem and leaves has already dissipated. Kuprian and Neuner, unpublished.
<img alt="Figure 8" src="/files/ftp_upload/52703/52703fig8.jpg" /> 
Figure 8.&#160;Hydrophobic barriers block extrinsic nucleation induced freezing of tomato (Solanum lycopersicon). (A) Hydrophobic kaolin-based material applied to tomato plants. (B&#8211;C). Reduced level of contact between the leaf surface and drops of liquid containing INA bacteria on coated (B) vs. uncoated (C) leaves. (D) Uncoated plant (right) undergoes an exothermic response associated with freezing of the plant while coated plant remains supercooled, and unfrozen at approximately -6 &#176;C. This figure has been modified from Wisniewski et al.34

Watch this Scientific Journal Video about The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants at JoVE.com

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).

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

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Enhancement of the Initial Growth Rate of Agricultural Plants by Using Static Magnetic Fields

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing seeds of Garden Balsam (Impatiens balsamina), Mizuna (Brassica rapa var. japonica), Komatsuna (Brassica rapa var. perviridis), and Mescluns (Lepidium sativum). We applied magnets under the culture dish. During the 4 days of application, we observed that the stem and root length increased. The group subjected to magnetic field treatment (n = 10) showed a 1.4 times faster rate of growth compared with the control group (n = 11) in a total of 8 days (p &#60;0.0005). This rate is 20% higher than that reported in previous studies. The tubulin complex lines did not have connecting points, but connecting points occur upon the application of magnets. This shows complete difference from the control, which means abnormal arrangements. However, the exact cause remains unclear. These results of growth enhancement of applying magnets suggest that it is possible to enhance the growth rate, increase productivity, or control the speed of germination of plants by applying static magnetic fields. Also, magnetic fields can cause physiological changes in plant cells and can induce growth. Therefore, stimulation with a magnetic field can have possible effects that are similar to those of chemical fertilizers, which means that the use of fertilizers can be avoided.

The goal of this protocol is to demonstrate the acceleration of the initial growth rate of plants by applying static magnetic fields with no external energy.

Electronic devices and high-voltage wires induce magnetic fields. A magnetic field of 1,300-2,500 Gauss (0.2 Tesla) was applied to Petri dishes containing ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus area. Biomass capacitance plays an integral role in the avoidance of hydraulic impairment to transpiration. However, high temporal resolution measurements of dynamic changes in the hydraulic capacitance of large trees are rare. Here, we present procedures for the calibration and use of capacitance sensors, typically used to monitor soil water content, to measure the volumetric water content in trees in the field. Frequency domain reflectometry-style observations are sensitive to the density of the media being studied. Therefore, it is necessary to perform species-specific calibrations to convert from the sensor-reported values of dielectric permittivity to volumetric water content. Calibration is performed on a harvested branch or stem cut into segments that are dried or re-hydrated to produce a full range of water contents used to generate a best-fit regression with sensor observations. Sensors are inserted into calibration segments or installed in trees after pre-drilling holes to a tolerance fit using a fabricated template to ensure proper drill alignment. Special care is taken to ensure that sensor tines make good contact with the surrounding media, while allowing them to be inserted without excessive force. Volumetric water content dynamics observed via the presented methodology align with sap flow measurements recorded using thermal dissipation techniques and environmental forcing data. Biomass water content data can be used to observe the onset of water stress, drought response and recovery, and has the potential to be applied to the calibration and evaluation of new plant-level hydrodynamics models, as well as to the partitioning of remotely sensed moisture products into above- and belowground components.

The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees.

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

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

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Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures