Cryo-Scanning Electron Microscopy to Study the Freezing Behavior of Plant Tissues.

A cryo-scanning electron microscope (cryo-SEM) is a valuable tool for observing bulk frozen samples to monitor freezing responses of plant tissues and cells. Here, the essential processes of a cryo-SEM to observe freezing behaviors of plant tissue cells are described.

Freeze-substitution transmission electron microscopy of gentian shoot tips cryopreserved at ultra low temperatures.

Transmission electron microscopy (TEM) combined with freeze substitution was employed to examine the ultrastructure of cells of gentian shoot tips cooled to the ultra-low temperature of slush nitrogen and liquid nitrogen. When shoot tips were cooled in ultra-low temperature without plant vitrification solution 2 (PVS2) treatment, massive ice formation was observed throughout the cells, indicating that severe injury occurred during cooling. In contrast, when shoot tips were treated with PVS2 and subsequently cooled to ultra- low temperatures, no ice crystals were observed in the cells.

Mechanism of freezing resistance in eco-dormant birch buds under winter subzero temperatures.

Maximum freezing resistance is a component of winter survival and is associated with the eco-dormant state. Differential thermal analysis (DTA) has shown that changes of the freezing response of the dormant buds depend not only on species and bud type, but also on cooling rates. In order to clarify the freezing adaptation at the cellular level of eco-dormant buds in Japanese white birch, birch buds cooled at a rate of 0.

Supercooling-Promoting (Anti-ice Nucleation) Substances.

Studies on supercooling-promoting substances (SCPSs) are reviewed introducing name of chemicals, experimental conditions and the supercooling capability (SCC) in all, so far recognized, reported SCPSs and results of our original study are presented in order to totally show the functional properties of SCPSs which are known in the present state. Many kinds of substances have been identified as SCPSs that promote supercooling of aqueous solutions in a non-colligative manner by reducing the ice nucleation capability (INC) of ice nucleators (INs). The SCC as revealed by reduction of freezing temperature (°C) by SCPSs differs greatly depending on the INs.

Anti-Ice Nucleating Activity of Surfactants against Silver Iodide in Water-in-Oil Emulsions.

Various water-soluble substances are known as anti-ice nucleating agents (anti-INAs), which inhibit heterogeneous ice nucleation initiated by ice nucleating agents (INAs). Among them, several surfactants are reportedly effective as anti-INAs especially against silver iodide (AgI), which is a typical inorganic INA that induces heterogeneous ice nucleation at relatively high temperatures. In this study, the anti-ice nucleating activities of seven surfactants were examined in emulsified surfactant solutions containing AgI particles.

Anti-ice nucleating activity of polyphenol compounds against silver iodide.

Freeze-avoiding organisms survive sub-zero temperatures without freezing in several ways, such as removal of ice nucleating agents (INAs), production of polyols, and dehydration. Another way is production of anti-ice nucleating agents (anti-INAs), such as has been reported for several antifreeze proteins (AFPs) and polyphenols, that inhibit ice nucleation by inactivating INAs. In this study, the anti-ice nucleating activity of five polyphenol compounds, including flavonoid and tannin compounds of both biological and synthetic origin, against silver iodide (AgI) was examined by measuring the ice nucleation temperature in emulsified polyphenol solutions containing AgI particles.

Cryo-scanning electron microscopy to study the freezing behavior of plant tissues.

A cryo-scanning electron microscope (cryo-SEM) is a valuable tool for observing bulk frozen samples to monitor freezing responses of plant tissues and cells. Here, essential processes of a cryo-SEM to observe freezing behaviors of plant tissue cells are described.

Analysis of supercooling activities of surfactants.

Supercooling-promoting activities (SCAs) of 25 kinds of surfactants including non-ionic, anionic, cationic and amphoteric types were examined in solutions (buffered Milli-Q water, BMQW) containing the ice nucleation bacterium (INB) Erwinia ananas, silver iodide (AgI) or BMQW alone, which unintentionally contained unidentified ice nucleators, by a droplet freezing assay. Most of the surfactants exhibited SCA in solutions containing AgI but not in solutions containing the INB E. ananas or BMQW alone.

Analysis of supercooling activity of tannin-related polyphenols.

Based on the discovery of novel supercooling-promoting hydrolyzable gallotannins from deep supercooling xylem parenchyma cells (XPCs) in Katsura tree (see Wang et al. (2012) [38]), supercooling capability of a wide variety of tannin-related polyphenols (TRPs) was examined in order to find more effective supercooling-promoting substances for their applications. The TRPs examined were single compounds including six kinds of hydrolyzable tannins, 11 kinds of catechin derivatives, two kinds of structural analogs of catechin and six kinds of phenolcarboxylic acid derivatives, 11 kinds of polyphenol mixtures and five kinds of crude plant tannin extracts.

Roles of cell walls and intracellular contents in supercooling capability of xylem parenchyma cells of boreal trees.

The supercooling capability of xylem parenchyma cells (XPCs) in boreal hardwood species differs depending not only on species, but also season. In this study, the roles of cell walls and intracellular contents in supercooling capability of XPCs were examined in three boreal hardwood species, Japanese beech, katsura tree and mulberry, whose supercooling capability differs largely depending on species and season. XPCs in these species harvested in winter and summer were treated by rapid freezing and thawing (RFT samples) or by RFT with further washing (RFTW samples) to remove intracellular contents from XPCs in order to examine the roles of cell walls in supercooling.

Freezing activities of flavonoids in solutions containing different ice nucleators.

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 μL droplets at 0.2 °C/min by a droplet freezing assay.

Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in Cercidiphyllum japonicum.

Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified.

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees.

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-D-glucopyranoside (K3Glc), kaempferol 7-O-β-D-glucopyranoside (K7Glc) and quercetin 3-O-β-D-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators.

Response to dehydration and irrigation in invasive and native saplings: osmotic adjustment versus leaf shedding.

To clarify the mechanism underlying successful invasion by tree species into xeric sites on Japan's Bonin Islands, we compared the water use of an alien species, Psidium cattleianum, which is rapidly expanding on ridge sites with shallow soil, with that of a native species, Trema orientalis. We hypothesized that there is a trade-off between leaf shedding with low cavitation resistance (frequent xylem cavitation plus refilling ability) and leaf osmotic adjustment with high cavitation resistance (cessation of xylem cavitation plus canopy leaf retention), indicating contrasting strategies for drought tolerance and water use in semi-arid regions. We examined leaf turnover, leaf gas exchange, leaf water potential and water distribution in stem xylem conduits using cryo-scanning electron microscopy for the saplings of both species under three cycles of artificial drought and sudden pulse irrigation.

Endoplasmic reticulum-localized small heat shock protein that accumulates in mulberry tree (Morus bombycis Koidz.) during seasonal cold acclimation is responsive to abscisic acid.

With seasonal changes, several proteins accumulate in the endoplasmic reticulum (ER)-enriched fraction in the bark of mulberry tree (Morus bombycis Koidz.). Results of partial amino acid sequence analysis in our previous study suggested that one of these proteins is the ER-localized small heat shock protein (sHSP), designated 20-kD winter-accumulating protein (WAP20).

Analysis of supercooling-facilitating (anti-ice nucleation) activity of flavonol glycosides.

Deep supercooling xylem parenchyma cells (XPCs) of katsura tree (Cercidiphyllum japonicum) contain four kinds of flavonol glycosides with high supercooling-facilitating (anti-ice nucleation) activities. These flavonol glycosides have very similar structures, but their supercooling-facilitating activities are very different. In this study, we analyzed the supercooling-facilitating activities of 12 kinds of flavonol glycosides in order to determine the chemical structures that might affect supercooling-facilitating activity.

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi).

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells.

Improved cryopreservation by diluted vitrification solution with supercooling-facilitating flavonol glycoside.

The effect of kaempferol-7-O-glucoside (KF7G), one of the supercooling-facilitating flavonol glycosides which was originally found in deep supercooling xylem parenchyma cells of the katsura tree and was found to exhibit the highest level of supercooling-facilitating activity among reported substances, was examined for successful cryopreservation by vitrification procedures, with the aim of determining the possibility of using diluted vitrification solution (VS) to reduce cryoprotectant toxicity and also to inhibit nucleation at practical cooling and rewarming by the effect of supplemental KF7G. Examination was performed using shoot apices of cranberry and plant vitrification solution 2 (PVS2) with dilution. Vitrification procedures using the original concentration (100%) of PVS2 caused serious injury during treatment with PVS2 and resulted in no regrowth after cooling and rewarming (cryopreservation).

Effects of light condition after simulated acid snow stress on leaves of winter wheat.

Winter plants regrow after freeze-thawing in acidic meltwater from the acid-snow layer in early winter or early spring. In this study, the responses of cold-acclimated wheat seedlings to different light conditions during the regrowth period after simulated acid snow (SAS) stress were investigated. After freeze-thawing in sulfuric acid (SAS stress) of pH 2.

Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity.

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near -40 degrees C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling.

Gene expression associated with increased supercooling capability in xylem parenchyma cells of larch (Larix kaempferi).

Xylem parenchyma cells (XPCs) in larch adapt to subfreezing temperatures by deep supercooling, while cortical parenchyma cells (CPCs) undergo extracellular freezing. The temperature limits of supercooling in XPCs changed seasonally from -30 degrees C during summer to -60 degrees C during winter as measured by freezing resistance. Artificial deacclimation of larch twigs collected in winter reduced the supercooling capability from -60 degrees C to -30 degrees C.

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells.

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb.

Larch (Larix kaempferi) xylem parenchyma cells respond to subfreezing temperature by deep supercooling.

In previous studies, xylem parenchyma cells (XPCs) in the boreal softwood species larch, which has thick and rigid walls similar to those of XPCs in boreal hardwood species, were shown to respond to subfreezing temperature by deep supercooling during summer but change their freezing behavior to extracellular freezing during winter. In this study, we re-examined freezing behavior of XPCs in larch by observation of deep etching of frozen samples as well as observation of re-warmed samples after freezing using a cryo-scanning electron microscope. The results showed that XPCs in larch adapts to subfreezing temperature by deep supercooling throughout all seasons.

High accumulation of soluble sugars in deep supercooling Japanese white birch xylem parenchyma cells.

Seasonal changes in the accumulation of soluble sugars in extracellular freezing cortical parenchyma cells and deep supercooling xylem parenchyma cells in Japanese white birch (Betula platyphylla var. japonica) were compared to identify the effects of soluble sugars on the mechanism of deep supercooling, which keeps the liquid state of water in cells under extremely low temperatures for long periods. Soluble sugars in both tissues were analyzed by high-performance liquid chromatography (HPLC), and the concentrations of sugars in cells were estimated by histological observation of occupancy rates of parenchyma cells in each tissue.