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. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

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 °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. Crude xylem extracts from katsura tree ( Cercidiphyllum japonicum ), of which XPCs exhibited deep supercooling to −40 °C, were prepared by methanol extraction. The crude extracts were purified by liquid–liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8–9.0 °C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3- O - β -glucoside (Q3G), kaempferol-7- O - β -glucoside (K7G), 8-methoxykaempferol-3- O - β -glucoside (8MK3G) and kaempferol-3- O - β -glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.     

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. 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.     

Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in <Emphasis Type="Italic">Cercidiphyllum japonicum</Emphasis>

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,β-d-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-β-d-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-β-d-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-β-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.     

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. All of the flavonol glycosides tested showed supercooling-facilitating activity, although the magnitudes of activity differed among the compounds. It was clear that the combination of the position of attachment of the glycosyl moiety, the kind of attached glycosyl moiety and the structure of aglycone determined the magnitude of anti-ice nucleation activity. However, there is still some ambiguity preventing the exact identification of features that affect the magnitude of supercooling-facilitating activity.     

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-β-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.     

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.     

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.     

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. As an approach to clarify the mechanisms underlying the change in supercooling capability of larch XPCs, genes expressed in association with increased supercooling capability were examined. By differential screening and differential display analysis, 30 genes were found to be expressed in association with increased supercooling capability in XPCs. These 30 genes were categorized into several groups according to their functions: signal transduction factors, metabolic enzymes, late embryogenesis abundant proteins, heat shock proteins, protein synthesis and chromatin constructed proteins, defence response proteins, membrane transporters, metal-binding proteins, and functionally unknown proteins. All of these genes were expressed most abundantly during winter, and their expression was reduced or disappeared during summer. The expression of all of the genes was significantly reduced or disappeared with deacclimation of winter twigs. Interestingly, all but one of the genes were expressed more abundantly in the xylem than in the cortex. Eleven of the 30 genes were thought to be novel cold-induced genes. The results suggest that change in the supercooling capability of XPCs is associated with expression of genes, including genes whose functions have not been identified, and also indicate that gene products that have been thought to play a role in dehydration tolerance by extracellular freezing also have a function by deep supercooling.     

766. CERCIDIPHYLLUM MAGNIFICUM – Systematic placement and fossil history of Cercidiphyllum Siebold & Zuccarini

Cercidiphyllum Sieb. & Zucc. comprises two very similar living species, Cercidiphyllum japonicum Sieb. & Zucc. and Cercidiphyllum magnificum (Nakai) Nakai, both commonly referred to as the katsura tree. The systematic placement and fossil history of Cercidiphyllum are described, and aspects of its cultivation and conservation are discussed. Cercidiphyllum magnificum is illustrated.     

Vulnerability to cavitation differs between current‐year and older xylem: non‐destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse‐porous species

Development of xylem embolism during water stress in two diffuse‐porous hardwoods, Katsura (Cercidiphyllum japonicum) and Japanese white birch (Betula platyphylla var. japonica), was observed non‐destructively under a compact magnetic resonance imaging (MRI) system in addition to conventional quantitation of hydraulic vulnerability to cavitation from excised stem segments. Distribution of white and dark areas in MR images corresponded well to the distribution of water‐filled/embolized vessels observed by cryo‐scanning electron microscopy in both species. Water‐filled vessels were observed in MR images as white areas in Katsura and as white dots in Japanese white birch, respectively, and embolisms could be detected as a change to dark areas. The increase in the relative embolized area (REA: %) in the cross‐sectional area of total xylem during water stress, which was estimated from the binarized MR images, was consistent with the hydraulic vulnerability curves of these species. From the non‐destructive MRI observations, cavitation induced by water stress was shown to develop earlier in 1‐ or 2‐year‐old xylem than in the current‐year xylem in both species; that is, the vulnerability to cavitation differs between vessels in the current‐year xylem and those in older annual rings.     

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. Relative and equilibrium melting points of parenchyma cells were measured by differential thermal analysis and cryoscanning electron microscopy, respectively. In both xylem and cortical parenchyma cells, amounts of sucrose, raffinose and stachyose increased in winter, but amounts of fructose and glucose exhibited little change throughout the entire year. In addition, no sugars were found to be specific for either tissue. Combined results of HPLC analyses, histological observation and melting point analyses confirmed that the concentration of sugars was much higher in xylem cells than in cortical cells. It is thought that the higher concentration of soluble sugars in xylem cells may contribute to facilitation of deep supercooling in xylem cells by depressing the nucleation temperature.     

Chromosome-level genome assembly of Japanese chestnut (Castanea crenata Sieb. et Zucc.) reveals conserved chromosomal segments in woody rosids

Japanese chestnut (Castanea crenata Sieb. et Zucc.), unlike other Castanea species, is resistant to most diseases and wasps. However, genomic data of Japanese chestnut that could be used to determine its biotic stress resistance mechanisms have not been reported to date. In this study, we employed long-read sequencing and genetic mapping to generate genome sequences of Japanese chestnut at the chromosome level. Long reads (47.7 Gb; 71.6× genome coverage) were assembled into 781 contigs, with a total length of 721.2 Mb and a contig N50 length of 1.6 Mb. Genome sequences were anchored to the chestnut genetic map, comprising 14,973 single nucleotide polymorphisms (SNPs) and covering 1,807.8 cM map distance, to establish a chromosome-level genome assembly (683.8 Mb), with 69,980 potential protein-encoding genes and 425.5 Mb repetitive sequences. Furthermore, comparative genome structure analysis revealed that Japanese chestnut shares conserved chromosomal segments with woody plants, but not with herbaceous plants, of rosids. Overall, the genome sequence data of Japanese chestnut generated in this study is expected to enhance not only its genetics and genomics but also the evolutionary genomics of woody rosids.     

Supercooling of xylem ray parenchyma cells in tropical and subtropical hardwood species

 The freezing behavior of xylem ray parenchyma cells in several woody species, Ficus elastica, F. microcarpa, Mangifera indica, Hibiscus Rosa-sinensis, and Schefflera arboricola, that are native to non-frost tropical and subtropical zones, was investigated by differential thermal analysis (DTA), cryo-scanning electron microscopy (cryo-SEM) and freeze-replica electron microscopy. Although profiles after DTA did not exhibit clear evidence of supercooling in the xylem ray parenchyma cells, electron microscopy revealed that the majority of xylem ray parenchyma cells in all of the woody species examined were supercooled to around –10°C upon freezing temperatures and were not frozen extracellularly. It seems likely that DTA failed to reveal the low temperature exotherm (LTE), that is produced by breakdown of supercooling in the xylem ray parenchyma cells as a consequence of the overlap between the high temperature exotherm and the LTE in each case. The xylem ray parenchyma cells in these woody species were very sensitive to dehydration, and supercooling had, to some extent, a protective effect against freezing injury. It is suggested that the capacity for supercooling of xylem ray parenchyma cells of tropical and subtropical woody species might be the result of inherent structural characteristics, such as rigid cell walls and compact xylem tissues, rather than the result of positive adaptation to freezing temperatures. The present and previous results together indicate that the responses of xylem ray parenchyma cells in a wide variety of hardwood species to freezing temperatures can be explained as a continuum, the specifics of which depend upon the temperatures of the growing conditions. Received: 24 January 1997 / Accepted: 13 May 1997     

CHANGES IN THE ULTRASTRUCTURE OF XYLEM PARENCHYMA CELLS OF PEACH (PRUNUS PERSICA) AND RED OAK (QUERCUS RUBRA) IN RESPONSE TO A FREEZING STRESS

An ultrastructural investigation was conducted of xylem parenchyma cells of peach (Prunus persica [L.] Batsch.) cv. Harbrite and red oak (Quercus rubra L.) in response to a freezing stress. Freezing curves of xylem tissues, as determined by differential thermal analysis, were used to predict temperatures at which both living and dead cells would be observed. Tissues were exposed to low temperatures (-15 to -35 C) and fixed in a frozen state at -10C and at thawing. Current models of the freezing behavior of supercooled plant cells suggest that xylem parenchyma cells behave as individual water droplets. This implies that cells are unresponsive to the presence of low temperature and extracellular ice until internal nucleation triggers lethal, intracellular freezing. For these reasons, deep supercooling has been described as an avoidance mechanism. Results of this study confirmed earlier reports that xylem parenchyma cells freeze as individuals or in small groups. Individual cells, however, did not exhibit a neutral response. Instead, a range of responses was observed that included internal and external vesiculation, deep invaginations of the plasma membrane, and the formation of electron-dense deposits external to the plasmalemma. In general, our observations suggested that the cells responded to a dehydrative stress. Results are discussed in context of the biophysical data associated with deep supercooling phenomena and compared to responses of cells that exhibit extracellular freezing.     

Platypus quercivorus ambrosia beetles use leaf volatiles in host selection

Although chemical volatiles emitted from host and non-host trees have been suggested as important cues for bark and ambrosia beetles, their responses to leaf volatiles is poorly understood. The oak ambrosia beetle, Platypus quercivorus (Murayama) (Coleoptera: Curculionidae), is a vector for the fungus that causes Japanese oak wilt. Using a Y-tube olfactometer, we tested the behavioral response of P. quercivorus to leaf volatiles emitted from four host trees – Quercus crispula Blume, Quercus serrata Murray, Quercus salicina Blume, and Castanea crenata Sieb. & Zucc. (all Fagaceae) – and two non-host trees, Fagus crenata Blume (Fagaceae) and Cryptomeria japonica D. Don (Cupressaceae). A flight mill was used to evaluate the effect of flight on the behavioral response to leaf volatiles. The bioassays were repeated 10× before and 10× after flight in the flight mill for each of the 54 individual beetles. Leaf volatile components were analyzed using gas chromatography–mass spectrometry. The bioassay results supported our hypothesis: P. quercivorus was attracted by the leaf volatiles of hosts and was deterred by the leaf volatiles of non-hosts. The behavioral response of P. quercivorus to the leaf volatiles was stronger after flight. Males had a stronger behavioral response than females to leaf volatiles. The leaf volatile chemical profile of the non-host C. japonica differed from the profile of the host plants. However, the chemical profile of the non-host F. crenata was similar to the profile of the hosts. Our findings provide insight into the functions of leaf chemical volatiles in the interaction of P. quercivorus with its hosts and non-hosts and may help improve the control of P. quercivorus and Japanese oak wilt.     

Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls

Summary By cryo-scanning electron microscopy we examined the effects of the organization of the cell walls of xylem ray parenchyma cells on freezing behavior, namely, the capacity for supercooling and extracellular freezing, in various softwood species. Distinct differences in organization of the cell wall were associated with differences in freezing behavior. Xylem ray parenchyma cells with thin, unlignified primary walls in the entire region (all cells inSciadopitys verticillata and immature cells inPinus densiflora) or in most of the region (mature cells inP. densiflora and all cells inP. pariflora var.pentaphylla) responded to freezing conditions by extracellular freezing, whereas xylem ray parenchyma cells with thick, lignified primary walls (all cells inCrytomeria japonica) or secondary walls (all cells inLarix leptolepis) in most regions responded to freezing by supercooling. The freezing behavior of xylem ray parenchyma cells inL. leptolepis changed seasonally from supercooling in summer to extracellular freezing in winter, even though no detectable changes in the organization of cell walls were apparent. These results in the examined softwood species indicate that freezing behavior of xylem ray parenchyma cells changes in parallel not only with clear differences in the organization of cell walls but also with subtle sub-electron-microscopic differences, probably, in the structure of the cell wall.     

New Diterpenes of Torreya nucifera Sieb. et Zucc.

Three new labdane type diterpenes; 4-epiagathadiol (named kayadiol), 18-hydroxymanool (named torreferol), 18-hydroxy-13-epimanool (named 13-epitorreferol), were isolated from the non-steam-volatile fraction of leaves of Torreya nucifera Sieb. et Zucc. (Taxaceae, Japanese name “Kaya”).

This is the first reported isolation these three diterpenes in a natural source.     

Radial patterns of xylem sap flow in non-, diffuse- and ring-porous tree species

We investigated radial patterns of sap flux density and wood properties in the sapwood of young loblolly pine (Finns taeda L.), mature white oak (Quercus alba L.) and sweetgum (Liquidambar styraciflua L.), which represent three major classes of wood anatomy: non-porous (coniferous), ring-porous and diffuse-porous. Radial measurements of xylem sap flux density were made in sections of xylem extending to 20 mm and 20–40 mm from the cambium. These measurements were compared with measurements of the relative water content (R_s) and sapwood specific gravity (ρ_r) of corresponding radial sections. In both hardwood species, sap flow differences were rarely significant between the two depth intervals. In pine, a 59% reduction in daily sap flux density from outer to inner sapwood was found. This could not be accounted for by a 3% drop in R_s; rather, an accompanying 9% reduction in ρ_r indicated a transition between the depth intervals from mature to juvenile sapwood, and is the probable cause of the lower flux rate in the inner xylem of pine.