Poplar leaf abscission through induced chlorophyll breakdown by Mg-dechelatase.

Chlorophyll breakdown is observed during senescence. The first step in chlorophyll breakdown is the removal of central Mg by Mg-dechelatase. This reaction is the rate-limiting step in the chlorophyll breakdown pathway. We evaluated the effect of induced chlorophyll breakdown on abscission through the removal of Mg by Mg-dechelatase. Poplar transformants carrying the dexamethasone-inducible Mg-dechelatase gene were prepared using the Arabidopsis Stay-Green1 cDNA. When leaves were treated with dexamethasone, chlorophyll was degraded, photosynthetic capacity was reduced, and an abscission zone was formed, resulting in leaf abscission. In addition, ethylene, which plays an important role during senescence, was produced in this process. Thus, chlorophyll breakdown induces the phenotype in the same way as commonly observed during leaf senescence. This study suggests a physiological role of chlorophyll breakdown in the leaf abscission of deciduous trees. Furthermore, this study shows that the dexamethasone-inducible gene expression system is an available option for deciduous tree studies.

Freezing resistance and behavior of winter buds and canes of wine grapes cultivated in northern Japan.

In high-latitude regions, the cold hardiness of buds and canes of grapevine is important for budburst time and yield in the next season. The freezing resistance of buds and canes sampled from six wine grapes currently cultivated in Hokkaido, Japan, all of them grown from autumn to winter, was investigated. A significant difference between the cultivars in their freezing resistance was detected in the buds harvested in winter. In addition, outstanding differences in the lower temperature exotherms (LTE) related to the supercooling ability of tissue cells happened in the winter buds, and there is a close relationship between freezing resistance and LTE detected in the winter buds. This suggests that the supercooling ability of tissue cells in winter buds is strongly related to the freezing resistance. However, detailed electron microscopy exposed that the differences in freezing resistance among cultivars appeared in freezing behavior of leaf primordium rather than apical meristem. This indicated that as the water mobility from the bud apical meristem to the spaces around the cane phloem progressed, the slightly dehydrated cells improved the supercooling ability and increased the freezing resistance.

Cryo-scanning electron microscopy reveals that supercooling of overwintering buds of freezing-resistant interspecific hybrid grape 'Yamasachi' is accompanied by partial dehydration.

Dormant compound buds of grapevines adapt to subfreezing temperatures through a freezing avoidance mechanism. One still-unclear question, however, is whether supercooled water in primordial cells of dormant grape buds are partially dehydrated under subfreezing temperatures. In this study, we used differential thermal analysis (DTA) and cryo-scanning electron microscopy (cryo-SEM) to look for partial dehydration of primordial cells of the freezing-resistant interspecific hybrid cultivar 'Yamasachi'. According to DTA, the freezing temperature of supercooled water in primary buds was not significantly affected by cooling rates between 2 and 5 °C/h; however, maintaining the bud temperature at -15 °C for 12 h followed by cooling at a rate of 5 °C/h depressed the freezing temperature. As revealed by cryo-SEM observation, many wrinkles were present on inner surfaces of walls and outer surfaces of plasma membranes of leaf primordial cells in dormant buds frozen to -15 °C. These results suggest the existence of partial dehydration in dormant-bud primordial cells under subfreezing temperatures. The apparent absence of extracellular ice crystals in bud primordial tissues under subfreezing temperatures suggests that Yamasachi dormant buds adapt to subfreezing temperatures by extraorgan freezing. When we coated primary buds with silicone oil to inhibit freeze dehydration of primordial cells, the freezing temperature of buds was slightly but significantly increased. This result suggests that the partial dehydration of cells promotes bud supercooling capability and has an important role in the freezing adaptation mechanism of grapevines.

Combination of the phosphodiesterase 10A inhibitor, MR1916 with risperidone shows additive antipsychotic-like effects without affecting cognitive enhancement and cataleptic effects in rats.

AIM: Phosphodiesterase 10A (PDE10A) inhibitors not only have antipsychotic-like effects but also cause cognitive enhancement without affecting extrapyramidal side effects in rodents, suggesting that PDE10A may be a novel approach for the treatment of schizophrenia. However, how a combination of PDE10A inhibitor with a currently available antipsychotic drug, risperidone contributes to the effect of each compound in rats remains unclear. The purpose of the present study was to examine the combination effects of MR1916 with a currently available antipsychotic drug, risperidone, in rats. METHODS: We examined the combination effects of the PDE10A inhibitor, MR1916 with risperidone on conditioned avoidance response (CAR) to assess antipsychotic-like effects in rats. We also examined them on catalepsy as extrapyramidal side effects and novel object recognition test in cognitive functions in rats. RESULTS: MR1916 (0.025-0.2 mg/kg, p.o.) and risperidone (0.75-6 mg/kg, p.o.) alone attenuated the CAR in a dose-dependent manner. The combination of MR1916 (0.025 mg/kg, p.o.) with risperidone (0.75 mg/kg, p.o.) significantly enhanced the attenuation of CAR without increasing the escape failure response. At the same dosage, the cataleptic effects were not enhanced by combined treatment of MR1916 with risperidone. Furthermore, the enhancement of object recognition memory induced by MR1916 (0.3 mg/kg, p.o.) was not affected by the combination with risperidone (0.75 mg/kg, p.o.). CONCLUSION: The combination of MR1916 with risperidone may have additive antipsychotic-like effects without affecting extrapyramidal side effects, and the cognitive-enhancing effect of MR1916 may not be interfered with the addition of risperidone.

Ameliorative effects of a phosphodiesterase 10A inhibitor, MR1916 on L-DOPA-induced dyskinesia in parkinsonian rats.

BACKGROUND: Dopamine replacement therapy using L-3,4-dihydroxyphenylalanine (L-DOPA) is a gold standard treatment in patients with Parkinson's disease (PD); however, chronic administration of L-DOPA causes excessive involuntary movements called L-DOPA-induced dyskinesia. Therefore, the novel pharmacological treatment is needed. METHODS: We examined the antidyskinetic effect of a phosphodiesterase 10A (PDE10A) inhibitor, MR1916 and a currently available antidyskinetic drug, amantadine in unilateral 6-OHDA lesioned rats exhibited stably dyskinesia after chronic administration of L-DOPA. We also examined the influence of MR1916 and amantadine on the improvement of forelimb akinesia induced by L-DOPA using stepping test in unilateral 6-OHDA lesioned rats. RESULTS: MR1916 (0.03‒0.3 mg/kg, po) reduced L-DOPA-induced dyskinesia in a dose-dependent manner and showed significant effects at doses of 0.1 and 0.3 mg/kg, while amantadine (40 mg/kg, sc) had no remarkable effects. Neither MR1916 (0.03‒0.3 mg/kg, po) nor amantadine (40 mg/kg, sc) affected the antiparkinsonian effects induced by L-DOPA in unilateral 6-OHDA lesioned rats. CONCLUSIONS: These results indicate that MR1916 specifically reduces L-DOPA-induced dyskinesia without affecting the antiparkinsonian effect of L-DOPA in parkinsonian rats.

Mechanism of Overwintering in Trees.

Boreal trees possess very high freezing resistance, which is induced by short-day length and low temperatures, in order to survive severe subzero temperatures in winter. During autumn, cooperation of photoreceptors and circadian clock system perceiving photoperiod shortening results in growth cessation, dormancy development, and first induction of freezing resistance. The freezing resistance is further enhanced by subsequent low temperature during seasonal cold acclimation with concomitant changes in various morphological and physiological features including accumulation of sugars and late embryogenesis abundant proteins. The mechanism of adaptation to freezing temperatures differs depending on the type of tissue in boreal trees. For example, bark, cambium, and leaf cells tolerate freezing-induced dehydration by extracellular freezing, whereas xylem parenchyma cells avoid intracellular freezing by deep supercooling. In addition, dormant buds in some trees respond by extraorgan freezing. Boreal trees have evolved overwintering mechanisms such as dormancy and high freezing resistance in order to survive freezing temperatures in winter.

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. While no single SCPS that affects homogeneous ice nucleation to reduce ice nucleation point has been found, many SCPSs have been found to reduce freezing temperatures by heterogeneous ice nucleation with a large fluctuation of SCC depending on the kind of heterogeneous IN. Not only SCPSs increase the degree of SCC (°C), but also some SCPSs have additional SCC to stabilize a supercooling state for a long term to stabilize supercooling against strong mechanical disturbance and to reduce sublimation of ice crystals. The mechanisms underlying the diverse functions of SCPSs remain to be determined in future studies.

Screening of plant resources with anti-ice nucleation activity for frost damage prevention.

Previous studies have shown that some polyphenols have anti-ice nucleation activity (anti-INA) against ice-nucleating bacteria that contribute to frost damage. In the present study, leaf disk freezing assay, a test of in vitro application to plant leaves, was performed for the screening of anti-INA, which inhibits the ice nucleation activity of an ice-nucleating bacterium Erwinia ananas in water droplets on the leaf surfaces. The application of polyphenols with anti-INA, kaempferol 7-O-ß-glucoside and (-)-epigallocatechin gallate, to the leaf disk freezing assay by cooling at -4--6 °C for 3 h, revealed that both the compounds showed anti-INAs against E. ananas in water droplets on the leaf surfaces. Further, this assay also revealed that the extracts of five plant leaves showed high anti-INA against E. ananas in water droplets on leaf surfaces, indicating that they are the candidate resources to protect crops from frost damage.

Pharmacological characterization of a novel potent, selective, and orally active phosphodiesterase 2A inhibitor, PDM-631.

Recently, we identified a novel phosphodiesterase 2A (PDE2A) inhibitor, PDM-631 ((S)-3-cyclopropyl-6-methyl-1-(1-(4-(trifluoromethoxy)phenyl)propan-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one). PDM-631 showed potent inhibitory activities for human and rat PDE2A with IC50 values of 1.5 and 4.2nM, respectively and more than 2000-fold selectivity against other phosphodiesterases. In rat studies, PDM-631 showed oral bioavailability and good brain penetration, and increased the cGMP levels in the cortex. These data indicate that PDM-631 is a potent, selective, orally active, and brain-penetrable PDE2A inhibitor. In behavioral studies using rat models, PDM-631 (3-30mg/kg) resulted in better discrimination between a novel object and a familiar one 48h after the acquisition phase in the novel object recognition test, thus indicating that PDM-631 increased object recognition memory. In contrast, PDM-631 did not attenuate the conditioned avoidance response at the same dose range (3-30mg/kg) in rats, indicating that PDM-631 did not show an antipsychotic-like effect. In test for extrapyramidal side effect, PDM-631 had no effect on catalepsy at the effective doses (10 and 30mg/kg) in the novel object recognition test, while haloperidol caused catalepsy at a dose of 3mg/kg. Our results suggest that PDM-631 is a good pharmacological tool that can be used to investigate the role of PDE2A and may have therapeutic potential for the treatment of cognitive impairments associated with schizophrenia and neurodegenerative disorders, without any extrapyramidal side effects.

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. Among previously reported anti-INAs (e.g., antifreeze proteins (AFPs), polyphenol compounds and synthetic polymers), a cationic surfactant used in this study, hexadecyltrimethylammonium bromide (C16TAB), showed the highest anti-ice nucleating activity against AgI. Based on the unique concentration-dependent dispersibility of AgI particles in C16TAB solution, the anti-ice nucleating activity of C16TAB must be caused by the adsorption of C16TAB molecules on AgI surfaces either as a monolayer or a bilayer depending on the C16TAB concentration.

Dopamine D1 signaling involvement in the effects of the phosphodiesterase 10A inhibitor, PDM-042 on cognitive function and extrapyramidal side effect in rats.

Inhibition of phosphodiesterase 10A (PDE10A) results in activation of a dopamine D1 receptor-mediated direct pathway in addition to a dopamine D2 receptor-mediated indirect pathway in the striatum. Therefore, PDE10A inhibitors could be novel therapeutics for schizophrenia, which differ from the currently available antipsychotics that directly block the dopamine D2 receptor. Previously, we found that a novel PDE10A inhibitor, PDM-042, had antipsychotic-like activity similar to currently available antipsychotics and minimal cataleptic effects in rats. The purpose of the present study was to examine the pharmacological effects of PDM-042 on cognitive function and extrapyramidal side effect. In addition, we aimed to examine whether these effects were mediated by activation of dopamine D1 signaling in rats. PDM-042 (1-3mg/kg) resulted in better discrimination of a novel object from a familiar one 48h after the acquisition trial, suggesting that PDM-042 increased object recognition memory. A dopamine D1 receptor antagonist, SCH23390 (0.1mg/kg), significantly blocked the enhancement of the object recognition memory induced by PDM-042 (3mg/kg) without affecting the recognition index by itself. We also found that the cataleptic effect of PDM-042 (1mg/kg) was significantly enhanced by SCH23390 (0.01-0.03mg/kg). These results indicate that PDM-042 has the potential to increase object recognition memory and that the cognitive enhancing and cataleptic effects of PDM-042 are mediated at least by activation of dopamine D1 signaling.

Pharmacological characterization of a novel potent, selective, and orally active phosphodiesterase 10A inhibitor, PDM-042 [(E)-4-(2-(2-(5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)vinyl)-6-(pyrrolidin-1-yl)pyrimidin-4-yl)morpholine] in rats: potential for the treatment of schizophrenia.

Recently, we identified a novel phosphodiesterase 10A (PDE10A) inhibitor, PDM-042 ((E)-4-(2-(2-(5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl)vinyl)-6-(pyrrolidin-1-yl)pyrimidin-4-yl)morpholine). PDM-042 showed potent inhibitory activities for human and rat PDE10A with IC 50 values of less than 1 nmol/L and more than 1000-fold selectivity against other phosphodiesterases. Tritiated PDM-042, [3H]PDM-042, had high affinity for membranes prepared from rat striatum with a Kd value of 8.5 nmol/L. The specific binding of [3H]PDM-042 was displaced in a concentration-dependent manner by PDM-042 and another structurally unrelated PDE10A inhibitor, MP-10. In rat studies, PDM-042 showed excellent brain penetration (striatum/plasma ratio = 6.3), occupancy rate (86.6% at a dose of 3 mg/kg), and good oral bioavailability (33%). These data indicate that PDM-042 is a potent, selective, orally active, and brain-penetrable PDE10A inhibitor. In behavioral studies using rat models relevant to schizophrenia, PDM-042 significantly antagonized MK-801-induced hyperlocomotion (0.1-0.3 mg/kg) without affecting spontaneous locomotor activity and attenuated the conditioned avoidance response (CAR) (0.3-1 mg/kg). In tests for adverse effects, PDM-042 had a minimal effect on catalepsy, even at a much higher dose (10 mg/kg) than the minimal effective dose (0.3 mg/kg) in the CAR. Furthermore, PDM-042 had no effect on prolactin release or glucose elevation up to 3 mg/kg, while risperidone increased prolactin release and olanzapine enhanced glucose levels at doses near their efficacious ones in the CAR. Our results suggest that PDM-042 is a good pharmacological tool that can be used to investigate the role of PDE10A and may have therapeutic potential for the treatment of schizophrenia.

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. The emulsified solutions eliminated the influence of contamination by unidentified INAs, thus enabling examination of the anti-ice nucleating activity of the polyphenols against AgI alone. Results showed that all five polyphenol compounds used here have anti-ice nucleating activities that are unique compared with other known anti-INAs, such as fish AFPs (type I and III) and synthetic polymers (poly(vinyl alcohol), poly(vinylpyrrolidone) and poly(ethylene glycol)). All five polyphenols completely inactivated the ice nucleating activity of AgI even at relatively low temperatures, and the first ice nucleation event was observed at temperatures between -14.1 and -19.4°C, compared with between -8.6 and -11.8°C for the fish AFPs and three synthetic polymers. These anti-ice nucleating activities of the polyphenols at such low temperatures are promising properties for practical applications where freezing should be prevented.

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. SCAs of many surfactants in solutions containing AgI were very high compared with those of previously reported supercooling-promoting substances. Cationic surfactants, hexadecyltrimethylammonium bromide (C16TAB) and hexadecyltrimethylammonium chloride (C16TAC), at concentrations of 0.01% (w/v) exhibited SCA of 11.8 °C, which is the highest SCA so far reported. These surfactants also showed high SCAs at very low concentrations in solutions containing AgI. C16TAB exhibited SCA of 5.7 °C at a concentration of 0.0005% (w/v).

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. The effects of these TRPs on freezing were examined by droplet freezing assays using various solutions containing different kinds of identified ice nucleators such as the ice nucleation bacterium (INB) Erwinia ananas, the INB Xanthomonas campestris, silver iodide and phloroglucinol as well as a solution containing only unintentionally included unidentified airborne ice nucleators. Among the 41 kinds of TRPs examined, all of the hydrolyzable tannins, catechin derivatives, polyphenol mixtures and crude plant tannin extracts as well as a few structural analogs of catechin and phenolcarboxylic acid derivatives exhibited supercooling-promoting activity (SCA) with significant differences (p>0.05) from at least one of the solutions containing different kinds of ice nucleators. It should be noted that there were no TRPs exhibiting ice nucleation-enhancing activity (INA) in all solutions containing identified ice nucleators, whereas there were many TRPs exhibiting INA with significant differences in solutions containing unidentified ice nucleators alone. An emulsion freezing assay confirmed that these TRPs did not essentially affect homogeneous ice nucleation temperatures. It is thought that not only SCA but also INA in the TRPs are produced by interactions with heterogeneous ice nucleators, not by direct interaction with water molecules. In the present study, several TRPs that might be useful for applications due to their high SCA in many solutions were identified.

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. RFT samples were also treated with glucose solution (RFTG samples) to examine roles of intracellular contents in supercooling. The supercooling capabilities of these samples were examined by differential thermal analysis after ultrastructural observation of XPCs by a cryo-scanning electron microscope to confirm effects of the above treatments. XPCs in RFTW samples showed a large reduction in supercooling capability to similar temperatures regardless of species or season. On the other hand, XPCs in RFTG samples showed a large increase in supercooling capability to similar temperatures regardless of species or season. These results indicate that although cell walls have an important role in maintenance of supercooling, change in supercooling capability of XPCs is induced by change in intracellular contents, but not by change in cell wall properties.

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. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT(50) between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4-4.2 °C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 °C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 °C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 °C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 °C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 °C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.

Cold acclimation in the moss Physcomitrella patens involves abscisic acid-dependent signaling.

Overwintering plants develop tolerance to freezing stress through a cold acclimation process by which the cells provoke internal protective mechanisms against freezing. The stress hormone abscisic acid (ABA) is known to increase freezing tolerance of plant cells, but its role in cold acclimation has not been determined. In this study, we used ABA-insensitive lines of the moss Physcomitrella patens to determine whether cold acclimation in bryophytes involves an ABA-dependent process. Two ABA-insensitive lines, both impaired in ABA signaling without showing ABA-induced stress tolerance, were subjected to cold acclimation, and changes in freezing tolerance and accumulation of soluble sugars and proteins were compared to the wild type. The wild-type cells acquired freezing tolerance in response to cold acclimation treatment, but very little increase in freezing tolerance was observed in the ABA-insensitive lines. Analysis of low-molecular-weight soluble sugars indicated that the ABA-insensitive lines accumulated sucrose, a major compatible solute in bryophytes, to levels comparable with those of the wild type during cold acclimation. However, accumulation of the trisaccharide theanderose and of specific LEA-like boiling-soluble proteins was very limited in the ABA-insensitive lines. Furthermore, analysis of cold-induced expression of genes encoding LEA-like proteins revealed that the ABA-insensitive lines accumulate only small amounts of these transcripts during cold acclimation. Our results indicate that cold acclimation of bryophytes requires an ABA-dependent signaling process. The results also suggest that cold-induced sugar accumulation, depending on the sugar species, can either be dependent or independent of the ABA-signaling pathway.

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. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2',5-tri-O-galloyl-α,ß-D-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-ß-D-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-ß-D-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5-4.5°C, at a concentration of 1 mg mL⁻¹. These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.

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-ß-D-glucopyranoside (K3Glc), kaempferol 7-O-ß-D-glucopyranoside (K7Glc) and quercetin 3-O-ß-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. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.