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A Tribute to Peter L. Steponkus |
A prospective student once asked me
what Pete Steponkus was like. "Half Rasputin, half Darth Vader,"
was my reply. Of course, I was joking, but to those who did not know
him, it might seem an apt description. Physically, he resembled
Rasputin (in Harley-Davidson clothes), and like Star Wars'
Vader, he often presented himself as a grim and imposing figure to
quaking, first-year graduate students. This demeanor, however, very
much depended on the day and situation, and was no doubt attributable
to the chronic pain he suffered from the degenerative disease
ankylosing spondylitis. Perhaps it was Pete's foreknowledge that the
quality of his life would decline precipitously and prematurely from
this disease that caused him to drive himself and his protegés so hard.
Pete, the son of a truck driver, grew up on "the wrong side of the
tracks in South Chicago," as he used to say with some pride. He was
the first member of his family to go to college, and one of the few in
his high school to do so. His interest in horticulture as a possible
career stemmed from a visit from the local mortician during his high
school's "career day." Once his education began in earnest, his
intellect and scholarship blossomed. Soon gone were his vague plans of
growing flowers for the mortuary business: He was on his way to
becoming one of the foremost cryobiologists of our time. An
inspirational teacher and a superb scholar, his masterful 1984 Annual Review of Plant Physiology contribution will be
required reading for decades to come.
I expect that Pete would have had little use for hagiography, and Pete
was no saint. His humor could be coarse, his behavior unrestrained. He
had little use for pomp or decorum or pretentiousness. He enjoyed being
the rebel, the troublemaker, and the thorn to his intellectual
"adversaries." He was a strong, unique presence in our staid and
stuffy academic world. Both science and I will miss him.
Here, I summarize some of Pete's more highly cited plant articles.
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Osmotic Contraction of Isolated Protoplasts |
During extracellular freezing, plant protoplasts
undergo dramatic contractions. Steponkus championed the use of
protoplasts to study the effects of freezing on the behavior of plasma
membranes. Gordon-Kamm and Steponkus (1984a)
demonstrated that isolated
rye (Secale cereale) leaf protoplasts contracted 50% in
response to osmotic stress. The plasma membrane, however, remains
smooth and non-pleated. Osmotic contraction is accompanied by the
endocytotic deletion of 40% of the plasma membrane and the production
of vesicles beneath the surface of the smooth plasma membrane. During
re-expansion of the protoplasts, these vesicles are not readily
reincorporated into the plasma membrane. Lysis generally results before
the protoplast regains its original volume.
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Acclimation Affects the Osmotic Behavior of
Protoplasts |
Videomicroscopy revealed that predominant form of
injury that occurred to isolated, nonacclimated (NA) rye protoplasts
during a freeze-thaw cycle to 
5°C was expansion-induced lysis: this was rarely observed in cold-acclimated (CA) protoplasts (Dowgert and
Steponkus, 1984). During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts, whereas exocytotic
extrusions of the plasma membrane were observed in CA
protoplasts. During thawing, the endocytotic vesicles of NA protoplasts
failed to be reincorporated into the plasma membrane, and the
protoplasts lysed. In contrast, the exocytotic vesicles formed during
the freezing of CA protoplasts were readily reincorporated into the plasma membraneduring thawing, and the protoplasts regained their original volume.
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Lamellar-to-HexagonalII (HII)
Phase Transitions |
Many phospholipids undergo a phase transition from a
lamellar to an (HII) phase at water contents of
approximately 20%. In pure phospholipid systems, the
phospholipids form long cylinders with the polar head
groups oriented in an aqueous core following the
dehydration-induced transition from a bilayer to the
HII phase. To investigate the possibility that
lamellar-to-HII phase transitions may be
involved in freezing injury in plants, Gordon-Kamm and Steponkus
(1984b) used freeze fracture to examine the changes in the morphology
of the plasma membrane of NA rye protoplasts following freeze-induced
dehydration. To further determine whether these changes were the
result of dehydration or cold, comparable osmotic manipulation and
supercooling studies were performed. Their studies revealed that
lamellarto-HII phase transitions do occur in
NA rye leaf protoplasts during freeze-induced dehydration, and that
this is attributable to dehydration rather than to cold per se. No
lamellar-to-HII phase transitions were observed
to occur in CA protoplasts subjected to freezing conditions.
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Effects of Acclimation on Lipid Composition of Plasma
Membranes |
The involvement of lipid alterations in cold acclimation had
a long and contentious history. In part, this controversy arose because
a majority of reports were based on correlative studies of changes in
lipid composition and cold hardiness that were based on analyses of
whole tissues or crude membrane preparations, rather the plasma
membrane per se. Lynch and Steponkus (1987) took advantage of two-phase
partitioning to isolate highly enriched plasma membrane fractions from
CA and NA rye seedlings. In short, the plasma membrane lipid
composition of purified plasma membrane fraction was shown to be
exceptional in containing high concentrations of glucocerebrosides, free sterols, and sterol derivatives, and relatively low concentrations of phospholipids. They further concluded that when the analysis is
carried out to the level of the lipid and the results expressed as mol
% of total lipid, the proportion of virtually every lipid component is altered during acclimation. Particularly marked following acclimation were the increases in the levels of free 
-sitosterol and
a doubling in the levels of those molecular species of
phosphatidylcholine and phosphatidylethanolamine bearing two
unsaturated acyl chains. Subsequent studies recorded the marked
differences that occur during acclimation in the lipid compositions of
the plasma membranes of spring oat, winter rye (Uemura and Steponkus,
1994), and Arabidopsis (Uemura et al., 1995).
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Fracture-Jump Lesions in Rye and Arabidopsis:
Freezing Injury to Acclimated Plants |
Webb and Steponkus (1993) studied freezing injury in leaves of NA
and CA rye by freeze-fracture electron microscopy. In protoplasts from
CA rye, freezing injury is associated not with the formation of the
inverted HII phase, but with localized deviations
in the fracture plane (fracture-jump lesions) between the plasma
membrane and closely appressed cytoplasmic membranes. At 10°C,
injury in NA leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the
plasma membrane, and by the frequent occurrence of the
HII phase. The HII phase
was not observed in leaves of CA rye frozen to 35°C. These findings
were later extended to CA oats (Webb et al., 1994) and Arabidopsis
(Uemura et al., 1995).
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Cold-Regulated Gene from Arabidopsis |
Artus et al. (1996) show that constitutive expression
of COR15a, a cold-regulated gene of Arabidopsis that encodes
a chloroplast-targeted polypeptide, enhances the in vivo freezing
tolerance of chloroplasts in NA plants by almost 2°C. Constitutive
expression of COR15a also affects the in vitro freezing
tolerance of protoplasts.
In a subsequent study, Steponkus et al. (1998) reported that the
increased freezing tolerance induced by the constitutive expression of
COR15a is the result of a decreased incidence of freeze-induced lamellar-to-HII phase transitions.
These phase transitions typically occur in regions where the plasma
membrane is brought into close apposition with the chloroplast
envelope as a result of freeze-induced dehydration. Moreover, the
mature polypeptide encoded by this gene, COR15am, increases the
lamellar-to-HII phase transition temperature of
dioleoylphosphatidylethanolamine and promotes formation of the lamellar
phase in a lipid mixture composed of the major lipid species that
comprise the chloroplast envelope. The authors propose that COR15am,
which is located in the chloroplast stroma, defers freeze-induced
formation of the HII phase to lower temperatures
(lower hydrations) by altering the intrinsic curvature of the inner
membrane of the chloroplast envelope.
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A Tribute to Peter...
Osmotic Contraction of Isolated...