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

Abstract

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 degrees 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.

Full text

Page 1

Instructions for use
Title
Anti-ice nucleation activity in xylem extracts from trees that
contain deep supercooling xylem parenchyma cells
Author(s)Kasuga, Jun; Mizuno, Kaoru; Arakawa, Keita; Fujikawa, Seizo
CitationCryobiology, 55(3): 305-314
Issue Date2007-12
Doc URL http://hdl.handle.net/2115/32347
Right
Type article (author version)
Additional
Information
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

Page 2

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

Jun Kasuga, Kaoru Mizuno, Keita Arakawa, Seizo Fujikawa*

Laboratory of Woody Plant Biology, Graduate School of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan
*Corresponding author. Address: Laboratory of Woody Plant Biology, Graduate School
of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
Fax: +81 011 706 3859. E-mail address: sfuji@for.agr.hokudai.ac.jp (S. Fujikawa)


















This study was supported by a grant from the Japan Society for the Promotion of
Science (17•9014 to J.K.) and a grant-in-aid for scientific research from the Ministry of
Education, Sports, Culture, Science and Technology of Japan (17380101 to S.F.).


1

Page 3

Abstract
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.

Key words: Supercooling; Cold acclimation; Boreal hardwood species; Anti-ice
nucleation substance; Heterogeneous ice nucleator


Introduction
Most trees have xylem parenchyma cells (XPCs) that adapt to subfreezing
temperatures by deep supercooling [3, 14, 15, 31, 32, 36, 41]. Water in XPCs that have
adapted to subfreezing temperatures by deep supercooling can be maintained in a liquid
state at very low temperatures for long periods. XPCs can survive in such a
supercooling state, but lethal intracellular freezing occurs if the temperature falls below
the limit of supercooling [14, 15]. Damage to XPCs caused by intracellular freezing

2

Page 4

may lead to death of entire trees [10, 35, 37]. Since the freezing resistance of XPCs that
adapt by deep supercooling is weaker than that of other tissue cells that adapt by
extracellular freezing [36, 37], the geographic distribution of trees in cold areas is
determined by the temperature limits of supercooling in XPCs [5, 7, 14, 16, 18].
The supercooling capability of XPCs changes greatly depending on the
environmental temperature. The temperature limits of supercooling in XPCs gradually
decrease from warm areas toward cold areas in parallel with latitudinal temperature
reduction, possibly reflecting evolutional cold acclimation [14, 18]. The temperature
limits of supercooling in XPCs also change depending on seasonal environmental
temperature as a result of seasonal cold acclimation and deacclimation [14, 25, 37, 46].
Furthermore, supercooling capability of XPCs is changed by artificial cold acclimation
and deacclimation of twigs in trees [22, 23]. Thus, trees adapt to environmental
temperature by changing the supercooling capability in their XPCs.
However, the mechanisms of deep supercooling in XPCs, especially the mechanisms
of the fluctuation of supercooling capability, are unclear. The mechanism of deep
supercooling in XPCs has been explained solely by the physical state of protoplasts as
an isolated water droplet [2, 17]. However, it is difficult to explain the cause of the
change in supercooling capability of XPCs only by such a physical state of water. On
the basis of a previous hypothesis, it has been suggested that the temperature limit of
supercooling corresponds to the temperature at which cell walls lose their barrier
property against penetration of extracellular ice. Our previous study, however, indicated
that walls of a wide variety of plant cells, even those of extracellular freezing cells in
trees, can inhibit penetration of extracellular ice [48].
Our recent studies have indicated that supercooling capability of XPCs is
significantly changed by release of intracellular substances and suggested that a variety
of intracellular substances might have important roles in the supercooling capability of
XPCs [26, 27]. We have hypothesized that there are intracellular substances in XPCs
that promote the supercooling capability of water. In the present study, therefore, we
tested this possibility. We prepared crude extracts from the xylem of several boreal
hardwood species that have deep supercooling XPCs and examined their effects on
supercooling of water droplets. The results indicated that these crude xylem extracts had
anti-ice nucleation activity which might be related to deep supercooling in XPCs.


3

Page 5

Materials and methods
Plant materials
Four to six-year-old twigs of 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.) were
harvested during winter (December to February) from adult trees growing in the campus
of Hokkaido University. The twigs were immediately placed on crushed ice and
transported to the laboratory. Xylem tissues, after the barks and piths had been removed,
were used as experimental materials.

Differential thermal analysis (DTA) for determination of supercooling capability in
XPCs
A block of fresh xylem tissue (300 mg) was connected to a copper-constantan
thermocouple (AUG #36) and wrapped with parafilm. Samples were placed in a deep
freezer (MDF-192, Sanyo Co., Ltd., Tokyo, Japan) equipped with a programmable
digital temperature controller (ES-100P, Tajiri Co., Ltd., Sapporo, Japan). The samples
were kept at 4°C for 60 min and then cooled at a rate of 0.2°C/min to –60°C, and the
differences between thermal responses of fresh samples and those of oven-dried
reference samples were recorded. All measurements were replicated three times.

Preparation of crude xylem extracts
Xylem tissues were cut into small pieces with a razor blade, frozen with liquid
nitrogen and ground into fine pieces with a mortar and pestle. Approximately 1 g of
xylem powder was extracted with 5 ml of 80% (v/v) ethanol overnight at room
temperature in the dark. The tissue debris was removed by centrifugation at 14,000 xg
for 10 min at 4°C. From the supernatants, ethanol and water were eliminated by
evaporation and the remnants were lyophilized. The lyophilized remnants were
redissolved in 200 µl of distilled water. The resultant suspensions were again
centrifuged at 14,000 xg for 10 min at 4°C and the supernatants were used as crude
xylem extracts. The crude xylem extracts were stored at –80°C until subsequent
examinations. The osmolarity of sample solutions was determined by using a vapor

4