Microscopic distribution of alkaloids in freeze-fixed stems of Phellodendron amurense.

Introduction: Phellodendron amurense Rupr. contains rich alkaloids, which have been extensively applied in clinical treatments for their various biological activities. However, detailed microscopic distribution and roles of such alkaloids in P. amurense stem still need to be clarified. Methods: In this study, the distribution of eight alkaloids in the transverse surface of freeze-fixed P. amurense stems in fall and summer has been visualized by cryo-time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM), which was found in living tissues with relative contents of different alkaloids varying with the position. In addition, the contents of these alkaloids quantified by high-performance liquid chromatography (HPLC) analysis suggested the seasonal variation from fall to the following summer. Results and discussion: Distribution of eight alkaloids in the freeze-fixed stems of P. amurense from fall and summer seasons has been visualized and assigned into specific living tissues, with relative contents varying in different positions with seasons, which suggested their possible roles in the physiological processes of the plant itself or plant responding to changes in the surrounding conditions. Conclusion: This study provided a significant basis for further discussion of the genes or enzymes involved in these processes, which will contribute to investigating biosynthetic pathways and specific in planta roles of alkaloids.

Direct protein delivery into intact plant cells using polyhistidine peptides.

Polyhistidine peptides (PHPs), sequences comprising only histidine residues (>His8), are effective cell-penetrating peptides for plant cells. Using PHP-fusion proteins, we aimed to deliver proteins into cultured plant cells from Nicotiana tabacum, Oryza sativa, and Cryptomeria japonica. Co-cultivation of cultured cells with fusion proteins combining maltose-binding protein (MBP), red fluorescent protein (RFP), and various PHPs (MBP-RFP-His8-His20) in one polypeptide showed the cellular uptake of fusion proteins in all plant cell lines. Maximum intracellular fluorescence was shown in MBP-RFP-His20. Further, adenylate cyclase (CyaA), a synthase of cyclic adenosine monophosphate (cAMP) activated by cytosolic calmodulin, was used as a reporter for protein delivery in living cells. A fusion protein combining MBP, RFP, CyaA, and His20 (MBP-RFP-CyaA-His20) was delivered into plant cells and increased intracellular fluorescence and cAMP production in all cell lines. The present study demonstrates that PHPs are effective carriers of proteins into the intracellular space of various cultured plant 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 0.2 °C min-1 and 5 °C day-1 were precisely examined by cryo-scanning electron microscopy (cryo-SEM). Freezing responses of floral dormant buds having female inflorescent primordia and leaf primordia with high-cold hardiness were assessed for extracellular freezing patterns by DTA. Cryo-SEM observation showed freezing of viscous solution filling intercellular spaces within buds and formation of extracellular ice in a random distribution within certain tissues, including green scales, leaf primordia and peduncles. The tissues producing extracellular ice had the common property that distinct intercellular spaces were present among cells having comparatively thick primary walls. In contrast, extracellular ice was not formed within flower primordium and parts of leaf primordium. These tissues had also the common property that no detectable intercellular spaces existed around the cells having thin primary walls. Cryo-SEM observation confirmed that all cells in tissues, regardless of whether extracellular ice was formed within tissues, and also regardless of differences in cooling rates, showed distinct cellular shrinkage by freezing. Recrystallization experiments by cryo-SEM confirmed that all freezable water in cells was eliminated by cooling at 0.2 °C min-1 at least to -30 °C. These results confirmed that all cells in birch buds responded to subzero temperatures through rapid equilibrium dehydration. In contrast to deep supercooling associated with extraorgan freezing of other freezing resistant buds of trees in an eco-dormant state, the mechanism of freezing resistance in eco-dormant birch buds is freezing adaptations by extracellular freezing.

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.

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 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.

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.

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. By slow cooling (5 degrees C/day) of dormant buds to -50 degrees C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to -20 degrees C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to -30 degrees C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to -50 degrees C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.