Commentaries in Plant Science. Volume 2

257 Pages·1981·4.488 MB·English

by  Harry Smith (Eds.)

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Commentaries in Plant Science
Volume 2
Edited by
HARRY SMITH
Department of Botany
The University of Leicester
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First edition 1981
British Library Cataloguing in Publication Data
Commentaries in plant science.
Vol. 2
1. Botany
581 QK45.2 80-41007
ISBN 0-08-025898-0
Printed in Great Britain by A. Wheaton & Co. Ltd., Exeter
PREFACE
Plant science is a rapidly developing subject of vital importance both for central, funda-
mental problems of modern biology and for the continued existence of mankind on this planet.
These seventeen Commentaries in Plant Science have been chosen to present topical and provoca-
tive "mini-reviews" of some of the more exciting recent developments in pure and applied plant
science, in a form which is easily digestible. The authors were encouraged to parade their own
viewpoints, to be constructively critical of existing work in their fields, and to be very selective
in their coverage of the literature. In eschewing the often uncritical, and always turgid, approach
of the fully comprehensive review, we have tried to provide entertaining and stimulating, as well as
instructive, reading. We hope that these articles will provoke discussion and experimentation
amongst research workers in plant science; aid the university and college teacher in coming to
grips with unfamiliar topics; and, above all, assist the hard-pressed student who requires topical
information, but is overwhelmed by the mass of primary literature being published.
The articles were published originally in the monthly issues of the current awareness
journal Current Advances in Plant Science between 1976 and 1980, on the recommendation of a
distinguished editorial board. For this compilation the Commentaries have been brought up to date
by their authors. The first volume of these Commentaries, published in 1977, was received with
enthusiasm and it is planned to produce further volumes of Commentaries, in due course.
ν
LIST OF CONTRIBUTORS
ALLEN, J.F., Botany School, South Parks Road, Oxford 0X1 3RA, U.K.
ASHMORE, M.R., Department of Plant Science, University of Leeds, Leeds LS2 9JT, U.K.
BARNETT, J.R., Plant Science Laboratories, University of Reading, Reading, Berks. RG6 2AS, U.K.
BURKE, MJ., Department of Horticulture, Colorado State University, Fort Collins, Colorado 80521,
U.S.A.
CHOLLET, R., University of Nebraska, Laboratory of Agricultural Biochemistry, Lincoln, NE 68583,
U.S.A.
DIGBY, J., Department of Biology, University of York, Heslington, York Y01 5DD, U.K.
DREW, M.C., Agricultural Research Council, Letcombe Laboratory, Wantage, 0X12 9JT, U.K.
EVANS, A.M., Department of Applied Biology, University of Cambridge, Cambridge, U.K.
FIRN, R.D., Department of Biology, University of York, Heslington, York Y01 5DD, U.K.
GEORGE, M.F., Department of Horticulture, Colorado State University, Fort Collins, Colorado 80521,
U.S.A.
GRIDLEY, H.E., Department of Applied Biology, University of Cambridge, Cambridge, U.K.
HART, J.W., Botany Department, University of Aberdeen, Aberdeen AB9 2UD, Scotland.
HIGGINS, T.J.V., CSIRO, Division of Plant Industry, Canberra 2601, Australia.
HOBSON, G.E., Glasshouse Crops Research Institute, Rustington, Littlehampton, West Sussex,
BN16 3PU,U.K.
HOPWOOD, D.A., John Innes Institute, Norwich, NR4 7UH, U.K.
HOWLETT, B.J., School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia.
INCOLL, L.D., Department of Plant Science, University of Leeds, Leeds LS2 9JT, U.K.
KNOX, R.B., School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia.
LONG, S.P., Department of Plant Science, University of Leeds, Leeds LS2 9JT, U.K.
PATHAK, M.D., The International Rice Research Inst., Los Banos, Laguna, Philippines.
RATHNAM-SHAGUTURU, Dow Chemical U.S.A., Central Research, New England Laboratory,
PO Box 400, Way land, MA 01778, U.S.A.
RAVEN, J.A., Department of Biological Sciences, University of Dundee, Dundee DD1 4HN,
Scotland.
SABNIS, D.D., Botany Department, University of Aberdeen, Aberdeen AB9 2UD, Scotland.
SAXENA, R.C., The International Rice Research Inst., Los Banos, Laguna, Philippines.
SMITH, F.A., Department of Biological Sciences, University of Dundee, Dundee DD1 4HN,
Scotland.
SPENCER, D.f CSIRO, Division of Plant Industry, Canberra 2601, Australia.
ST ACE, C.A., Botany Department, The University, Leicester LE1 7RH, U.K.
VASANTHE VITHANAGE, H.I.M., School of Botany, University of Melbourne, Parkville, Victoria
3052, Australia.
ix
1
THE OCCURRENCE OF DEEP SUPERCOOLING IN COLD HARDY PLANTS
2 3
MILON F. GEORGE and MICHAEL J. BURKE
Laboratory of Plant Hardiness, Department of Horticultural Science,
University of Minnesota, St. Paul, Minnesota 55108, U.S.A.
INTRODUCTION
It has long been accepted that cold hardy plants generally survive subfreezing temperatures
by tolerating extracellular freezing and associated cellular dehydration and that intracellular freezing
of supercooled cellular water is abnormal and does not occur in nature. Scarth and Levitt (33) demon-
strated that ice formation occurs first on cell walls outside the protoplasm and vacuole. Ice growth
under slow freezing conditions proliferates in intercellular spaces as water diffuses from the cell in
response to decreasing tissue temperature. Soluble concentration or melting point depression of the
cell sap keeps pace with ambient temperature so the cellular water does not freeze. Earlier micro-
scopic work by Weigand (39) in 1906, conducted in the field at temperatures near — 20°C, had shown
that in buds and twigs of many woody species ice crystals always were found in the intercellular
spaces of the tissue and that the cells were in a "more or less state of collapse". Even as early as 1860
the locus of ice formation had been observed to be intercellular (30). More recent work of Burke et
al. (3) and Gustaef at. (16) using pulsed nuclear magnetic resonance spectroscopy suggests that freez-
ing curves of plants which tolerate extracellular ice are very similar to those of ordinary salt solutions
and that the primary difference between hardy and non-hardy varieties is their ability to tolerate
cellular dehydration. Olien (24) has demonstrated that mechanical stress from ice formation is a
freezing vector as well as cellular dehydration.
Depending on the particular species, cold hardy plant tissues can tolerate extracellular
ice formation and cellular contraction to varying limits of low temperature. Siminovitch and Scarth
(34) found hardy cabbage epidermis survives only to approximately —10°C while Weiser (40) notes
that fully cold acclimated red osier dogwood bark and xylem can survive liquid nitrogen temperatures
(—196°C) with no apparent injury. The reader is referred to Levitt's (21) treatise on environmental
stress for detailed reviews of various proposed mechanisms dealing with low temperature injury in
plants which exhibit extracellular ice formation.
Although the overwhelming evidence of early hardiness research supported the concept of
extracellular freezing, a few examples were noted concerning lack of ice formation in plant parts at
1
These studies were supported in part by Grants to M.J.B. from the National Science Foundation (BMS 74-23137),
the Nerken Foundation and Minnesota Agricultural Experiment Station. Miscellaneous Journal Series Paper 1611 of
t2he Minnesota Agricultural Experiment Station.
Present address: Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg,
V3A 24061.
Present address: Department of Horticulture, Colorado State University, Fort Collins, Colorado 80521.
1
2 Milon F. George and Michael J. Burke
low temperature. Weigand (39) as stated above observed ice in twigs and buds during winter, but
also showed that certain living vegetative buds could be cooled to — 26.5°C before ice crystals were
observed. Dorsey and Strausbaugh (6) found ice in the bud scales and axis of dormant plum buds,
but not in the primordial region at —29.5°C. In neither case, however, did the researchers propose
that the tissues were in fact supercooled. Among the first to propose deep supercooling as a freezing
injury avoidance mechanism in plants were Tumanov and Krasavtsev (37) who observed low tempera-
ture freezing points or exotherms in oak, birch, fir, and pine branches. Tumanov and coworkers (38)
have also observed low temperature freezing points in cherry flower buds. Krasavtsev (18) suggests
that prolonged retention of supercooled water can lead to frost injury in marginally hardy trees such
as apple, the injury occurring from intracellular freezing of a trapped fraction of supercooled cellular
water at low temperature. Mild supercooling, less than 10°C has been proposed as an injury avoidance
mechanism in olive leaves (20) and some fruit buds (17, 23). Despite these few exceptions to extra-
cellular ice formation as the vehicle to avoidance of low temperature injury, Levitt (21) was justified
to conclude in 1972 that pronounced supercooling plays no major role in frost resistance of most
plants.
Recent evidence, however, suggests that deep supercooling may indeed play a significant
part in frost resistance of many plants. Work on overwintering floral primordia (9, 12, 15, 27, 36) and
hardwood xylem (4,10,11,13, 25, 26) of many woody species, to be discussed below, suggests that
these plant parts can only attain maximum cold hardiness near —40°C. Temperatures near — 40°C are
of physical significance since they are in the range of homogeneous ice nucleation temperatures for
supercooled aqueous solutions (28). The implications for the northern distribution of plants utilizing
supercooling as a frost avoidance mechanism are clear and are discussed further in the following
sections.
SUPERCOOLING IN AQUEOUS SOLUTIONS
A supercooled system is in a "metastable equilibrium" as described by Glasstone (14).
Actually most of the living systems to be described are in a transient non-equilibrium state where the
rate of decay of the state is exceedingly slow. Glasstone uses the term "metastable equilibrium" to
define an equilibrium state which is not the most stable state at a particular temperature. A metastable
system will undergo a spontaneous transition on the addition of the stable phase. When water is
cooled below its freezing point without ice formation it is in a metastable state. There is a theoretical
as well as empirical limit on the stability of supercooled water. This limit is referred to as the homo-
geneous ice nucleation temperature where spontaneous change from the metastable state (liquid) to
the stable state (ice) occurs. Many investigators have shown that very pure water droplets can be super-
cooled to low temperatures, but never below approximately — 40°C (2, 7, 19, 28). Frenkel's (8) hetero-
phase fluctuation theory for water, in which a distribution of constantly forming and reforming mole-
cular clusters (some ice-like) is proposed to exist in the liquid state, has been used as a model for
predicting the homogeneous nucleation temperature by nucleation theory. Above the homogeneous
nucleation temperature the distribution of clusters both in terms of mole fraction and size is such
that the rate of formation of critical size ice-like clusters capable of growing into macroscopic ice is
extremely slow. Water, in principle, may remain supercooled within 2 or 3°C of its homogeneous
nucleation point almost indefinitely. As noted by Rasmussen and MacKenzie (29), nucleation
theory indicates the primary reasons why the cluster distribution reaches an unstable point at the
homogeneous nucleation temperature. The relative number in terms of mole fraction and cluster size
increases rapidly at the nucleation point along with a continuing exponential decrease in the critical
cluster size, leading to rapid formation of a critical size ice nucleus. Although nucleation theory pre-
The Occurrence of Deep Supercooling in Cold Hardy Plants 3
diets the fundamental character of homogeneous ice formation, it does not predict precisely the
experimental temperature at which ice nucleation occurs. This primarily results from the difficult
task of assigning an accurate value to the surface tension between the solid and liquid phases.
Fletcher's (7) theoretical derivation fits to a reasonable degree most experimental observations on
supercooled water where the homogeneous nucleation temperature is -38° ± 1 C.
Homogeneous ice nucleation is predicated on the concept that the water contains no
heterogeneous nucleators. Foreign surfaces and suspended insoluble particles promote hetero-
DEIONIZDE DISTILLDE WATRE
+60|
+50| H-20
-30 >
+20j -50
-HO 60
Ο
-60 -50 -40 -30 -20 -I0
REFERENCE TEMPERATURE, °C
Β
-80j
\ETHYLENE GLYCOL
\PEG
-70
•y
NaC,3vvk %
U -60 \oS\
ο UARNEDA CnV
GLYCEROL-^ΝΛΚ^
W Λ
-50 3\0\ -0 \
-40
-35 ι ι I
-20 -I5 -I0 -5
Figure 1. A. Thermogram for emulsified deionized distilled water. B. Homogeneous nucleation
temperature (Tn) versus melting temperature of aqueous solutions (Tm). Rasmussen and
MacKenzie (28).
4 Milon F. George and Michael J. Burke
geneous nucleation at higher temperatures. Increase in the nucleation temperature depends on the
size and surface properties of the impurities (7) Solutes, however, generally increase the super-
cooling necessary for crystallization (28). It might be asked how water can be studied at tempera-
tures near its homogeneous nucleation temperature (—38°C) since heterogeneous nucleators, even
in very small amounts, will nucleate supercooled water at higher temperatures. One of the most
successful methods for supercooling water to the homogeneous nucleation temperature is by
homogenizing water into droplets in hydrocarbon solvents (28). Some of the water droplets freeze
due to ice nucleation from heterogeneous impurities or from spontaneous crystallization but this is an
almost undetectably small fraction of the droplets at temperatures above — 38°C (Figure 1). A tristea-
rate surfactant included in the emulsion isolates any heterogeneous ice nucleation above —38°C so
that ice propagation does not occur By using this method, Rasmussen and MacKenzie (28) have
shown that solutes including glucose, urea, polyvinylpyrrolidone, NaCI, glycerol, ethylene, ethylene
glycol, and NH4F depress the nucleation temperature proportionately more than the melting point
depression (Figure 1).
Although it would be reasonable to expect the living cell to provide many sites for
heterogeneous nucleations, MacKenzie eta/. (22) indicate that ice-like structures do not generally
exist in the cell and that the cytoplasm behaves much like a dilute aqueous solution. Their suggestion
is based on experiments where individual yeast cells survived supercooling to —37°C.
DEEP SUPERCOOLING IN PLANT REPRODUCTIVE PARTS
SJight supercooling in fruit buds, especially at the first stages of anthesis, has been
suggested as an injury avoidance mechanism in peach, but the extent of supercooling is generally less
than —10°C (17, 23). Tumanov eta/. (38) were the first to assign low temperature injury in reproduc-
tive parts to freezing of supercooled water. In calorimetric experiments conducted on cherry flower
buds they found rapid freezing events between —20° and — 30°C which were associated with injury to
the tissues. In 1971, Graham (15) made an extensive study of low temperature freezing points in
Rhododendron flower buds. He found that cold hardy Rhododendron floral primordia did not freeze
at temperatures near —2°C as did the bud scales and stem axis to which they are attached, but froze
at temperatures as low as —43°C. Primordia invariably survived low temperatures if they did not
freeze, but death occurred to a primordium if it froze. Low temperature freezing points have since
been observed in floral primordia of blueberry (36) and several Prunus species. (27)
Differential thermal analysis (d.t.a.), differential scanning calorimetry (d.s.c.) and nuclear
magnetic resonance spectroscopy (n.m.r.) have been used to study water in floral primordia of
Rhododendron during the dormant winter months (9, 12). A d.t.a. recording of freezing a whole
flower bud (Figure 2) clearly displays the major freezing events or exotherms associated with ice forma-
tion. The large first exotherm results from freezing of water in the bud scales while succeeding exo-
therms are a consequence of ice nucleation in each individual primordium. Bud scale exotherms remain
constant over the winter season but primordia freeze at lower average temperatures in midwinter than
in fall or spring. Plants which exhibit extracellular ice formation follow the general rule that freezing
and thawing occur at the same temperature. Cooling and thawing analysis of an excised primordium
by d.s.c, d.t.a. and n.m.r. indicate the presence of only liquid or supercooled water and no ice from
0°C to the freezing point. Upon subsequent warming the primordium thaws gradually with a melting
point near —2°C in a manner analogous to a solution containing approximately one mole of an
osmotically active solution. This is not a characteristic of a melting point depression or eutectic freez-
ing phenomenon which are well defined solid-liquid equilibrium processes that have the same
The Occurrence of Deep Supercooling in Cold Hardy Plants 5
MARCH I
Figure 2. D.t.a. recordings of freezing profiles of whole azalea buds cooled at 8.5°C/hr. On 1
March, the bud is a typical midwinter one. On 16 April the convergence of exotherms indicates
loss of hardiness in early spring. Exotherm 1 corresponds to freezing of water in the bud scales
and stem axis upon which the primordia are attached. Exotherms of type 2 indicate freezing of
an individual primordium. The number of primordia exotherms generally equals the number of
primordia in the bud at a cooling rate of 8.5°C/hr. Each division on the differential response
scale equals approximately 1.4°C. George etal. (9)
crystallization and melting temperatures. Heat of fusion measurements find that heat released when a
primordium freezes is close to the value expected for supercooled water (12). From a physical stand-
point the fraction of water freezing at subzero temperatures in floral primordia appears to be super-
cooled water.
Microscopic observation (12) of primordial leaves mounted in fluid fluorocarbon and
cooled to subfreezing temperatures show that crystallization takes place randomly at a point in the
leaf and then rapidly grows throughout the entire leaf. This sudden freezing of supercooled tissue
involves intracellular freezing as observed by the sudden darkening of the cells. Siminovitch and
Scarth (34) have shown that intracellular freezing in cortical cells of Catalpa and Cornus trees,
brought on artificially by rapid cooling, causes visible mechanical disintegration and death to the
protoplasm. In almost all cases reported, intracellular freezing has been found to be fatal in plant
cells. If the primordial leaf is mounted in water and nucleated near 0°C, the tissues freeze at higher
temperatures than if mounted and cooled in fluorocarbon. Ice growth proceeds gradually throughout
the leaf and although the small cell size and experimental conditions prevented close observation, the
mode of freezing was probably at least in part extracellular. No barrier to ice growth seems to exist
between cells. Since in the whole bud primordia are surrounded by stem and bud scale tissues con-
taining ice, there must exist a physical barrier to ice propagation between this ice and the supercooled
primordial tissues. Freezing and associated injury to the floral primordia in winter cereals has been
shown to depend somewhat on the ability of the vascular tissue to stop the advance of the freezing
boundary (35). A study of whole Rhododendron buds cooled at different rates has found that
primordia freeze at higher temperatures when cooled faster than approximately 8°C/hr supporting
the concept of a physical barrier to ice growth between primordia and the scales and stem axis (9).
Some underlying physical property of the primordia must also exist to prevent sublimation of the
supercooled liquid water to the ice in the nearby tissues through the vapor phase. Regardless of the