Freezing stress damage and growth viability in Vaccinium macrocarpon Ait. bud structures.

After a freezing event, it can be challenging to extrapolate levels of freezing damage to plant growth viability based on the presence or absence of symptoms in specific bud tissues. This study investigated the relationship between freezing damage in terminal buds during ecodormancy and their viability during the subsequent growing season. We identified the bud structure that best explained this relationship, and developed a model to explain the changes in bud cold hardiness. Vertical shoots (uprights) of Vaccinium macrocarpon Ait. were sampled in central Wisconsin during Spring of 2018 and 2019. Sets of uprights with terminal buds were subjected to controlled freezing tests, followed by either visual freeze damage evaluation or assessment of shoot viability by growth assays. We determined the Browning Lethal-Temperature50 (BLT50 ), as temperature for 50% damage (tissue browning) at each bud structure, and Growth Lethal-Temperature50 (GLT50 ) temperature where 50% reduction in growth viability occurred. Two models were constructed to explain: (1) bud structure damage and growth viability, and (2) GLT50 's seasonal changes, representing the cold hardiness variations, and environmental factors. The correlation between the BLT50 and GLT50 values was closest for the bud scales and bud axis, indicating the better correspondence between levels of freezing damage with the impact on the growth potential. In addition, the latter was also the most suitable candidate for modeling due to easier damage evaluation. The freezing stress damage of the bud axis explained comparatively best the resulting growth viability. Seasonal changes in GLT50 were best explained by temperature indices based on daily minimum and on maximum temperatures over 10-day periods. However, among the model components, daily maximum temperatures had the greatest influence on V. macrocarpon cold hardiness changes during ecodormancy.

Acquisition of Freezing Tolerance in Vaccinium macrocarpon Ait. Is a Multi-Factor Process Involving the Presence of an Ice Barrier at the Bud Base.

Bud freezing survival strategies have in common the presence of an ice barrier that impedes the propagation of lethally damaging ice from the stem into the internal structures of buds. Despite ice barriers' essential role in buds freezing stress survival, the nature of ice barriers in woody plants is not well understood. High-definition thermal recordings of Vaccinium macrocarpon Ait. buds explored the presence of an ice barrier at the bud base in September, January, and May. Light and confocal microscopy were used to evaluate the ice barrier region anatomy and cell wall composition related to their freezing tolerance. Buds had a temporal ice barrier at the bud base in September and January, although buds were only freezing tolerant in January. Lack of functionality of vascular tissues may contribute to the impedance of ice propagation. Pith tissue at the bud base had comparatively high levels of de-methyl-esterified homogalacturonan (HG), which may also block ice propagation. By May, the ice barrier was absent, xylogenesis had resumed, and de-methyl-esterified HG reached its lowest levels, translating into a loss of freezing tolerance. The structural components of the barrier had a constitutive nature, resulting in an asynchronous development of freezing tolerance between anatomical and metabolic adaptations.

A device for the controlled cooling and freezing of excised plant specimens during magnetic resonance imaging.

BACKGROUND: Investigating plant mechanisms to tolerate freezing temperatures is critical to developing crops with superior cold hardiness. However, the lack of imaging methods that allow the visualization of freezing events in complex plant tissues remains a key limitation. Magnetic resonance imaging (MRI) has been successfully used to study many different plant models, including the study of in vivo changes during freezing. However, despite its benefits and past successes, the use of MRI in plant sciences remains low, likely due to limited access, high costs, and associated engineering challenges, such as keeping samples frozen for cold hardiness studies. To address this latter need, a novel device for keeping plant specimens at freezing temperatures during MRI is described. RESULTS: The device consists of commercial and custom parts. All custom parts were 3D printed and made available as open source to increase accessibility to research groups who wish to reproduce or iterate on this work. Calibration tests documented that, upon temperature equilibration for a given experimental temperature, conditions between the circulating coolant bath and inside the device seated within the bore of the magnet varied by less than 0.1 °C. The device was tested on plant material by imaging buds from Vaccinium macrocarpon in a small animal MRI system, at four temperatures, 20 °C, - 7 °C, - 14 °C, and - 21 °C. Results were compared to those obtained by independent controlled freezing test (CFT) evaluations. Non-damaging freezing events in inner bud structures were detected from the imaging data collected using this device, phenomena that are undetectable using CFT. CONCLUSIONS: The use of this novel cooling and freezing device in conjunction with MRI facilitated the detection of freezing events in intact plant tissues through the observation of the presence and absence of water in liquid state. The device represents an important addition to plant imaging tools currently available to researchers. Furthermore, its open-source and customizable design ensures that it will be accessible to a wide range of researchers and applications.

Freezing stress survival mechanisms in Vaccinium macrocarpon Ait. terminal buds.

Plants' mechanisms for surviving freezing stresses are essential adaptations that allow their existence in environments with extreme winter temperatures. Although it is known that Vaccinium macrocarpon Ait. buds can acclimate in fall and survive very cold temperatures during the winter, the mechanism for survival of these buds is not known. The main objective of this study was to determine which of the two major mechanisms of freezing stress survival, namely, deep supercooling or freeze-induced dehydration, are employed by V. macrocarpon terminal buds. In the present study, no low-temperature exotherms (LTEs) were detected by differential thermal analysis. Furthermore, a gradual reduction of relative liquid water content in the inner portions of buds during magnetic resonance imaging (MRI) scans performed between 0 and -20 °C (where no damage was detected in controlled freezing tests (CFT)) indicates these buds may not deep supercool. The higher ice nucleation activity of outer bud scales and the appearance of large voids in this structure in early winter, in conjunction with the MRI observations, are evidence supportive of a freeze-induced dehydration process. In addition, the presence of tissue browning in acclimated buds as a result of freezing stress was only observed in CFT at temperatures below -20 °C, and this damage gradually increased as test temperatures decreased and at different rates depending on the bud structure. Ours is the first study to collect multiple lines of evidence to suggest that V. macrocarpon terminal buds survive long periods of freezing stress by freeze-induced dehydration. Our results provide a framework for future studies of cold hardiness dynamics for V. macrocarpon and other woody perennial species and for the screening of breeding populations for freezing stress tolerance traits.