Infrared thermography of in situ natural freezing and mechanism of winter-thermonasty in Rhododendron maximum.

Evergreen leaves of Rhododendron species inhabiting temperate/montane climates are typically exposed to both high radiation and freezing temperatures during winter when photosynthetic biochemistry is severely inhibited. Cold-induced "thermonasty," that is, lamina rolling and petiole curling, can reduce the amount of leaf area exposed to solar radiation and has been associated with photoprotection in overwintering rhododendrons. The present study was conducted on natural, mature plantings of a cold-hardy and large-leaved thermonastic North American species (Rhododendron maximum) during winter freezes. Infrared thermography was used to determine initial sites of ice formation, patterns of ice propagation, and dynamics of the freezing process in leaves to understand the temporal and mechanistic relationship between freezing and thermonasty. Results indicated that ice formation in whole plants is initiated in the stem, predominantly in the upper portions, and propagates in both directions from the original site. Ice formation in leaves initially occurred in the vascular tissue of the midrib and then propagated into other portions of the vascular system/venation. Ice was never observed to initiate or propagate into palisade, spongy mesophyll, or epidermal tissues. These observations, together with the leaf- and petiole-histology, and a simulation of the rolling effect of dehydrated leaves using a cellulose-based, paper-bilayer system, suggest that thermonasty occurs due to anisotropic contraction of cell wall cellulose fibers of adaxial versus abaxial surface as the cells lose water to ice present in vascular tissues.

Visualising the effect of freezing on the vascular system of wheat in three dimensions by in-block imaging of dye-infiltrated plants.

Infrared thermography has shown after roots of grasses freeze, ice spreads into the crown and then acropetally into leaves initially through vascular bundles. Leaves freeze singly with the oldest leaves freezing first and the youngest freezing later. Visualising the vascular system in its native 3-dimensional state will help in the understanding of this freezing process. A 2 cm section of the crown that had been infiltrated with aniline blue was embedded in paraffin and sectioned with a microtome. A photograph of the surface of the tissue in the paraffin block was taken after the microtome blade removed each 20 µm section. Two hundred to 300 images were imported into Adobe After Effects and a 3D volume of the region infiltrated by aniline blue dye was constructed. The reconstruction revealed that roots fed into what is functionally a region inside the crown that could act as a reservoir from which all the leaves are able to draw water. When a single root was fed dye solution, the entire region filled with dye and the vascular bundles of every leaf took up the dye; this indicated that the vascular system of roots was not paired with individual leaves. Fluorescence microscopy suggested the edge of the reservoir might be composed of phenolic compounds. When plants were frozen, the edges of the reservoir became leaky and dye solution spread into the mesophyll outside the reservoir. The significance of this change with regard to freezing tolerance is not known at this time. Thermal cameras that allow visualisation of water freezing in plants have shown that in crops like wheat, oats and barley, ice forms first at the bottom of the plant and then moves upwards into leaves through water conducting channels. Leaves freeze one at a time with the oldest leaves freezing first and then younger ones further up the stem freeze later. To better understand why plants freeze like this, we reconstructed a 3-dimensional view of the water conducting channels. After placing the roots of a wheat plant in a blue dye and allowing it to pull the dye upwards into leaves, we took a part of the stem just above the roots and embedded it in paraffin. We used a microtome to slice a thin layer of the paraffin containing the plant and then photographed the surface after each layer was removed. After taking about 300 images, we used Adobe After Effects software to re-construct the plant with the water conducting system in three dimensions. The 3D reconstruction showed that roots fed into a roughly spherical area at the bottom of the stem that could act as a kind of tank or reservoir from which the leaves pull up water. When we put just one root in dye, the entire reservoir filled up and the water conducting channels in every leaf took up the dye. This indicates that the water channels in roots were not directly connected to specific leaves as we had thought. When plants were frozen, the dye leaked out of the reservoir and spread into cells outside. Research is continuing to understand the significance of this change during freezing. It is possible that information about this effect can be used to help breeders develop more winter-hardy crop plants.

Cell wall modification by the xyloglucan endotransglucosylase/hydrolase XTH19 influences freezing tolerance after cold and sub-zero acclimation.

Freezing triggers extracellular ice formation leading to cell dehydration and deformation during a freeze-thaw cycle. Many plant species increase their freezing tolerance during exposure to low, non-freezing temperatures, a process termed cold acclimation. In addition, exposure to mild freezing temperatures after cold acclimation evokes a further increase in freezing tolerance (sub-zero acclimation). Previous transcriptome and proteome analyses indicate that cell wall remodelling may be particularly important for sub-zero acclimation. In the present study, we used a combination of immunohistochemical, chemical and spectroscopic analyses to characterize the cell walls of Arabidopsis thaliana and characterized a mutant in the XTH19 gene, encoding a xyloglucan endotransglucosylase/hydrolase (XTH). The mutant showed reduced freezing tolerance after both cold and sub-zero acclimation, compared to the Col-0 wild type, which was associated with differences in cell wall composition and structure. Most strikingly, immunohistochemistry in combination with 3D reconstruction of centres of rosette indicated that epitopes of the xyloglucan-specific antibody LM25 were highly abundant in the vasculature of Col-0 plants after sub-zero acclimation but absent in the XTH19 mutant. Taken together, our data shed new light on the potential roles of cell wall remodelling for the increased freezing tolerance observed after low temperature acclimation.

Using Pixel-Based Microscope Images to Generate 3D Reconstructions of Frozen and Thawed Plant Tissue.

Histological analysis of frozen and thawed plants has been conducted for many years but the observation of individual sections only provides a two-dimensional representation of a three-dimensional phenomenon. Currently available optical sectioning techniques for viewing internal structures in three dimensions are either low in resolution or the instrument cannot penetrate deep enough into the tissue to visualize the whole plant. Methods using higher resolution equipment are expensive and often require time-consuming training. In addition, conventional stains cannot be used for optical sectioning techniques. We present a relatively simple and less expensive technique using pixel-based (JPEG) images of conventionally stained histological sections of an Arabidopsis thaliana plant. The technique uses commercially available software to generate a 3D representation of internal structures.

Three-dimensional reconstruction of soybean nodules provides an update on vascular structure.

PREMISE OF THE STUDY: In many cases, the functioning of a biological system cannot be correctly understood if its physical anatomy is incorrectly described. Accurate knowledge of the anatomy of soybean [Glycine max (L.) Merril] nodules and its connection with the root vasculature is important for understanding its function in supplying the plant with nitrogenous compounds. Previous two-dimensional anatomical observations of soybean nodules led to the assumption that vascular bundles terminate within the cortex of the nodule and that a single vascular bundle connects the nodule to the root. We wanted to see whether these anatomical assumptions would be verified by digitally reconstructing soybean nodules in three dimensions. METHODS: Nodules were dehydrated, embedded in paraffin, and cut into 15 µm thick sections. Over 200 serial sections were stained with safranin and fast green, and then photographed using light microscopy. Images were digitally cleared, aligned, and assembled into a three-dimensional (3D) volume using the Adobe program After Effects. KEY RESULTS: In many cases, vascular bundles had a continuous connection around the nodules. The 3D reconstruction also revealed a dual vascular connection originating in the nodule and leading to the root in 22 of the 24 nodules. Of the 22 dual connections, 11 maintained two separate vascular bundles into the root with independent connections to the root vasculature. CONCLUSIONS: A more robust and complex anatomical pathway for vascular transport between nodules and root xylem in soybean plants is indicated by these observations and will contribute to a better understanding of the symbiotic relationship between soybean plants and nitrogen-fixing bacteria within the nodules.

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.

High-definition infrared thermography of ice nucleation and propagation in wheat under natural frost conditions and controlled freezing.

MAIN CONCLUSION: An extremely high resolution infrared camera demonstrated various freezing events in wheat under natural conditions. Many of those events shed light on years of misunderstanding regarding freezing in small grains. Infrared thermography has enhanced our knowledge of ice nucleation and propagation in plants through visualization of the freezing process. The majority of infrared analyses have been conducted under controlled conditions and often on individual organs instead of whole plants. In the present study, high-definition (1280 × 720 pixel resolution) infrared thermography was used under natural conditions to visualize the freezing process of wheat plants during freezing events in 2016 and 2017. Plants within plots were found to freeze one at a time throughout the night and in an apparently random manner. Leaves on each plant also froze one at a time in an age-dependent pattern with oldest leaves freezing first. Contrary to a common assumption that freezing begins in the upper parts of leaves; freezing began at the base of the plant and spread upwards. The high resolution camera used was able to verify that a two stage sequence of freezing began within vascular bundles. Neither of the two stages was lethal to leaves, but a third stage was demonstrated at colder temperatures that was lethal and was likely a result of dehydration stress; this stage of freezing was not detectable by infrared. These results underscore the complexity of the freezing process in small grains and indicate that comprehensive observational studies are essential to identifying and selecting freezing tolerance traits in grain crops.

Complex bud architecture and cell-specific chemical patterns enable supercooling of Picea abies bud primordia.

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to -50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D-reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell-specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl-like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At -18 °C, break-down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature-dependent freeze dehydration (-0.1 MPa K-1 ) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin-rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell-specific biochemical analysis.

Persistent Supercooling of Reproductive Shoots Is Enabled by Structural Ice Barriers Being Active Despite an Intact Xylem Connection.

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages (shoot elongation, flowering, fruiting) despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 µm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances (lignin, suberin, and cutin). A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 µm x 0.7 µm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris.

Visualizing surface area and volume of lumens in three dimensions using images from histological sections.

Visualizing the interior (lumen) of a tubular structure within tissue can provide a unique perspective on anatomical organization of the tissue. Portal tracts of the liver contain several vessels and ducts in various patterns of intertwining branches and are an example of such spaces. An inexpensive method, using light microscopy and a sample of conventionally stained canine livers, was used to colorize and allow visualization of the lumens of vessels within the portal tract in three dimensions. When the colour of the background was digitally cleared and the lumen filled with a solid colour, it was possible to measure areas and volumes of the portal vein, arteries, bile ducts and lymphatics. Significant differences between vessels and ducts across lobes and gender in control samples are discussed. Differences were also found between control and mixed breed dogs and between controls and a dog that died of accidental traumatic haemorrhage. These differences are discussed in relation to visualizing lumens using images generated from a light microscope. Vessels in plants such as xylem and continuously formed spaces resulting from ice formation are other examples where this technique could be applied.

Three-dimensional reconstruction of frozen and thawed plant tissues from microscopic images.

Histological analysis of frozen and thawed plants has been conducted for many years but the observation of individual sections provides only a 2-dimensional representation of a 3-dimensional phenomenon. Most techniques for viewing internal plant structure in three dimensions are either low in resolution or the instrument cannot penetrate deep enough into the tissue to visualize the whole plant. Techniques with higher resolution are expensive and equipment often requires time-consuming training. We present a relatively simple and less-expensive technique using pixel-based (JPEG) images of histological sections of an Arabidopsis thaliana plant and commercially available software to generate 3D reconstructions of internal structures. The technique has proven to work just as effectively for images from medical histology.

Histological analysis and 3D reconstruction of winter cereal crowns recovering from freezing: a unique response in oat (Avena sativa L.).

The crown is the below ground portion of the stem of a grass which contains meristematic cells that give rise to new shoots and roots following winter. To better understand mechanisms of survival from freezing, a histological analysis was performed on rye, wheat, barley and oat plants that had been frozen, thawed and allowed to resume growth under controlled conditions. Extensive tissue disruption and abnormal cell structure was noticed in the center of the crown of all 4 species with relatively normal cells on the outside edge of the crown. A unique visual response was found in oat in the shape of a ring of cells that stained red with Safranin. A tetrazolium analysis indicated that tissues immediately inside this ring were dead and those outside were alive. Fluorescence microscopy revealed that the barrier fluoresced with excitation between 405 and 445 nm. Three dimensional reconstruction of a cross sectional series of images indicated that the red staining cells took on a somewhat spherical shape with regions of no staining where roots entered the crown. Characterizing changes in plants recovering from freezing will help determine the genetic basis for mechanisms involved in this important aspect of winter hardiness.

Effectiveness of a theory-based risk reduction HIV prevention program for rural Vietnamese adolescents.

As of April 2003, 64,801 HIV cases have been documented in Vietnam (Policy Project 2003), 53.9 % of which are among individuals 20-29 years of age. Although HIV education efforts have increased, there remains a need for proven effective programs. We present findings from a randomized-controlled effectiveness trial of an HIV prevention program for adolescents 15-20 years. Four hundred eighty adolescents were randomized into control and intervention groups. Evaluation data were collected using the Vietnamese Youth Health Risk Behavior Instrument, including scales based on the protection motivation theory (PMT). Findings presented show significant differences in knowledge of severity and vulnerability of HIV/AIDS ( p < .05), perceptions of self-efficacy ( p < .001), and response efficacy for condom use ( p < .05) between control and intervention youth at immediate and 6-month postintervention. A significant difference was also found for response cost of condom use ( p < .05) at immediate postintervention. No significant difference was found for the construct of response cost at 6 months, and there were no significant differences for the constructs external and internal rewards. Reported engagement in vaginal sex (1.7%), or anal and/or oral sex (3.1%) was extremely low, and therefore changes in actual behaviors could not be accurately measured. However, intention to use condoms in possible future sexual encounters increased significantly ( p < .05) for the intervention youth compared to control youth between baseline (74/240, 30.8%) and both immediate postintervention (132/230, 57.4%), and six month follow-up (123/228: 53.9%). These data suggest the potential applicability of the PMT for HIV program development with non-Western adolescents but also point to the need for further studies on how constructs within behavioral theories might need to be modified in different sociocultural settings.