Plant Molecular Biology 35: 323–330, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Expression of a synthetic antifreeze protein in potato reduces electrolyte
release at freezing temperatures
James G. Wallis
, Hongyu Wang
& Daniel J. Guerra
Department of Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844, USA (
correspondence); present addresses:
Institute of Biological Chemistry, Washington State University, Pullman,
Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey
Received 29 January 1997; accepted in revised form 30 May 1997
Key words: transgenic Russet Burbank, cold tolerance
A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves
at freezing temperatures. The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the
extracytoplasmic space where ice crystallization ﬁrst occurs. The gene was introduced to Solanum tuberosum L.
cv. Russet Burbank by Agrobacterium-mediated transformation. Transformants were identiﬁed by PCR screening
and expression of the introduced protein was veriﬁed by immunoblot. Electrolyte-release analysis of transgenic
plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to
freezing. This is the ﬁrst identiﬁcation of a phenotype associated with antifreeze protein expression in plant tissue.
Abbreviations: AFP, antifreeze protein; PHA, phytohemagglutinin
All plants are susceptible to injury by cold and frost.
The damage caused by frost and cold has been thor-
ing the extent and causes of the damage has been
compiled . The speciﬁc mechanisms by which
frost kills plant cells are known. When temperature
decreases, ice formation on the epidermal surface is
promoted by the presence of ice-nucleating bacteria
presentthere .Typically, at about
C, ice form-
ation begins within the plant tissue, in the extracyto-
plasmicspacewherethe soluteconcentrationis lowest.
mic space withdraws water from the cytoplasm, desic-
cating cells and shrinking cellular membranes .
Althoughthe plasmalemmaforms a physicalbarrier to
the propagation of ice, if the temperature of the tissue
decreases to about
C, ice formation can pen-
etrate the cytoplasm, coincident with fracture of the
cell membrane and destruction of sub-cellular organ-
ization. Ice formation within the cytoplasm is lethal.
If, instead of decreasing, the temperature begins to
rise before ice penetrates the cytoplasm, cells may be
injuredby the disruption of membranesas they recover
from the shrunken, desiccated state. This expansion-
induced lysis may be the primary cause of freezing
injury [33, 34]. Throughout the cycle of desiccation,
membrane shrinkage, and thawing, electrolytes are
released from damaged cells [14, 25]. Plants have
developed a variety of defenses against low temper-
atures [4, 31]. At the cellular level, frost-hardy species
proline to reduce damage by desiccation. The physic-
al state of membrane lipids may be altered, with an
increase in fatty acid desaturation and decrease in ster-
ol and proteincontent. Finally, some cold-hardyplants
produce speciﬁc antifreeze proteins .
AFP inhibit ice growth and recrystallization. AFP
are effective in protecting against frost damage at con-
centrations several orders of magnitude lower than
colligative compounds [8, 3]. Proteins with speciﬁc
antifreeze properties were ﬁrst characterized in winter
ﬂounder (Pseudopleuronectes americanus) and short-
GR: 201001994, Pips nr. 142330 BIO2KAP
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