Blackwell Science, LtdOxford, UK

PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002

256June 2002
861
Light utilization and cold acclimation
L. V. Savitch et al.
10.1046/j.0016-8025.2002.00861.x
Original ArticleBEES SGML

Plant, Cell and Environment (2002) 25, 761771

Two different strategies for light utilization in

photosynthesis in relation to growth and cold acclimation
L. V. SAVITCH,2* E. D. LEONARDOS,3* M. KROL,2 S. JANSSON,1 B. GRODZINSKI,3 N. P. A. HUNER2 & G. QUIST1

1
Ume Plant Science Center, Department of Plant Physiology, Ume University, S-901 87 Ume, Sweden, 2Department of Plant
Sciences, The University of Western Ontario, London, ON, N6A 5B7 Canada and 3Department of Plant Agriculture, University
of Guelph, Guelph, ON, N1G 2W1 Canada

ABSTRACT Fo, instantaneous fluorescence with all PSII centres open in

Seedlings of Lodgepole pine (Pinus contorta L.) and winter
wheat (Triticum aestivum L. cv. Monopol) were cold accli-
mated under controlled conditions to induce frost hardi-
ness. Lodgepole pine responded to cold acclimation by
partial inhibition of photosynthesis with an associated par-
tial loss of photosystem II reaction centres, and a reduction
in needle chlorophyll content. This was accompanied by a
low daily carbon gain, and the development of a high and
sustained capacity for non-photochemical quenching of
absorbed light. This sustained dissipation of absorbed light
as heat correlated with an increased de-epoxidation of the
xanthophyll cycle pigments forming the quenching forms
antheraxanthin and zeaxanthin. In addition, the PsbS pro-
tein known to bind chlorophyll and the xanthophyll cycle
pigments increased strongly during cold acclimation of
pine. In contrast, winter wheat maintained high photosyn-
thetic rates, showed no loss of chlorophyll content per leaf
area, and exhibited a high daily carbon gain and a minimal
non-photochemical quenching after cold acclimation. In
accordance, cold acclimation of wheat neither increased the
de-epoxidation of the xanthophylls nor the content of the
PsbS protein. These different responses of photosynthesis
to cold acclimation are correlated with pine, reducing its
need for assimilates when entering dormancy associated
with termination of primary growth, whereas winter wheat
maintains a high need for assimilates as it continues to grow
and develop throughout the cold-acclimation period. It
appears that without evolving a sustained ability for con-
trolled dissipation of absorbed light as heat throughout the
winter, winter green conifers would not have managed to
adapt and establish themselves so successfully in the cold
climatic zones of the northern hemisphere.
Key-words: Pinus contorta; Triticum aestivum; cold acc-
limation; dormancy; evergreen; frost hardening; photo-
inhibition; photosynthesis; PsbS protein; xanthophyll cycle.
Abbreviations: A, antheraxanthin; Chl, chlorophyll; CP,
chlorophyllprotein complex; EPS, epoxidation state;
Correspondence: Gunnar quist. Fax: + 46 90 786 66 76; e-mail:
gunnar.oquist@plantphys.umu.se
*These authors contributed equally to the present study.
2002 Blackwell Science Ltd
the dark; Fm, maximum fluorescence with all PSII centres
closed; Fo, instantaneous fluorescence with all PSII cen-
tres open in the light; Fv/Fm, efficiency of open PSII reac-
tion centres in light; IRGA, infrared gas analyser; LHCII,
light-harvesting chlorophyllprotein complex II; Lhcb and
Lhca, light-harvesting pigmentprotein complex of PSII
and PSI, respectively; NCER, net carbon exchange rate;
NPQ, non-photochemical quenching; PSII, photosystem II;
PSII, quantum yield of PSII electron transport; Rd, dark
respiration; V, violaxanthin; Z, zeaxanthin.
INTRODUCTION
It has been known for a long time that photosynthesis pro-
vides the energy required for cold acclimation of plants
during the autumn, making them resistant to freezing win-
ter temperatures (Levitt 1980). However, the combined
exposure of plants to low temperatures and light as
required for frost hardening, increases the probability that
plants will succumb to partial inactivation of photosynthesis
due to photo-inhibition (quist, Gardestrm & Huner
2001). This may not be a major problem for deciduous spe-
cies that lose their leaves as the plants enter the stage of
winter dormancy in response to a shortened daylength, but
evergreen species such as Scots pine (Pinus sylvestris L.)
may suffer substantial photo-inhibition of photosynthesis
during the autumn months (Ottander, Campbell & quist
1995). In contrast, however, herbaceous winter annuals
such as winter cereals are much more resistant to low
temperature-induced photo-inhibition of photosynthesis
(quist & Huner 1993).
We have proposed earlier (Huner et al. 1993) that these
different autumnal responses of photosynthesis in conifers
and cereals, which both maintain their green foliage
throughout the autumn and winter months, must be related
to their different growth and developmental strategies
(Levitt 1980). Conifers such as Scots pine must enter a
short-day-dependent dormant growth state in the autumn
in order to attain maximum freezing tolerance. In contrast,
cereals such as winter rye are not dormant during cold accli-
mation but require growth and development at low temper-
atures to exhibit maximum freezing tolerance.
In a comparative study of Lodgepole pine (Pinus con-
torta L.) and winter wheat (Triticum aestivum L.), we now
761
762 L. V. Savitch et al.

demonstrate that the different growth and developmental consisted of four plexiglass plant-holding chambers com-
strategies exhibited by these two species during cold accli- puterized to analyse and control the environment of each
mation is well correlated with two totally different strate- chamber separately. The CO2 concentration was monitored
gies to utilize the photosynthetically absorbed light under and adjusted by an infrared gas analyser (IRGA; Analytical
prevailing low temperature conditions. Development Co., Hoddesdon, UK), while the chamber
was in an open mode. Preset CO2 concentrations were
maintained at 35 or 100 Pa by adding pure CO2 with a mass
MATERIALS AND METHODS
flow controller (MKS Instruments, Nepean, Ontario, Can-
Plant material and growth conditions ada). While measuring the CO2 exchange rate, the chamber
was in a closed mode. A second IRGA (LI-6262; Li-Cor
Winter wheat (Triticum aestivum L., cv. Monopol, Highland
Inc., Lincoln, NE, USA) monitored the CO2 concentration
Seeds, Blenheim, Canada) and seedlings of Lodgepole pine
due to photosynthesis and photorespiration. The net carbon
(Pinus contorta L.) were grown under controlled environ-
exchange rate (NCER) was calculated from the initial and
mental conditions. Winter wheat was grown in coarse
final CO2 concentrations, and from the pure CO2 injection
vermiculite in 7 cm plastic pots at a density of 10 plants
measurements as previously described by Dutton et al.
per pot. Seeds were germinated under a temperature
(1988). The CO2 release in the dark was used to determine
regime of 20/16 C (day/night) at a photon flux density
whole-plant dark respiration. Over a 24 h day/night period,
(PFD) of 250 mol m2 s1 (20 C/250 PFD) and a 16 h pho-
daily carbon gain of the whole plant was estimated by
toperiod. After 7 d, when the primary leaf had fully
recording the NCER during whole day/night cycles (Dut-
expanded, some of the seedlings were shifted to growth
ton et al. 1988). Whole-plant daily net carbon gain was cal-
conditions of 5 C/250 PFD for cold acclimation. Control
culated as: C = Cd Cn, where Cd is the daytime carbon gain
plants remained at the 20 C/250 PFD growth regime. Fully
and Cn is the night-time carbon loss. Daytime net carbon
expanded fourth leaves of 75-day-old, cold-acclimated and
gain (Cd) is the integrated NCER during the light period:
25-day-old 20 C grown plants were used for pigment anal-
m
ysis, gel electrophoresis and fluorescence measurements. At
Cd = ( NCERi xt i )
these stages, seedlings were considered to be of similar i =1
physiological age based on detailed growth kinetics (Hurry
where NCERi (e.g. mol C m2 s1) is the whole-plant net
& Huner 1991), and were used for whole-plant net CO2
photosynthetic rate over a period of ti, and m is the number
exchange measurements.
of NCER measurements. Night-time carbon loss (Cn) is the
Cold-acclimated and dark-adapted 1-year-old seedlings
integrated NCER during the dark period:
of Lodgepole pine were obtained from a pine nursery. To
k
initiate the second year growth, seedlings were transferred
Cn = ( NCER j xt j )
to the growth chamber with a temperature regime of j =1
25/15 C (day/night), 75% humidity, a PFD of 250 mol
where NCERj is the whole-plant dark respiration rate dur-
m2 s1 (25 C/250 PFD) and a 17 h photoperiod. After a
ing a period of tj and k is the number of NCER measure-
period of 6 weeks at 25 C/250 PFD, the second-year need-
ments. The rates of whole-plant dark respiration (Rd, mol
les were fully developed and considered as summer pine.
m2 s1) were calculated by averaging NCERj over the dark
At this stage the plants were transferred to a temperature
period.
regime of 15/10 C (day/night), 75% humidity, a PFD of
Measurements of whole-plant NCER for each environ-
250 mol m2 s1 (15 C/250 PFD) and an 8 h photoperiod.
mental challenge were made over 24 h periods with record-
After a period of 6 weeks at 15 C/250 PFD the plants were
ings every 6 min. Prior to NCER measurements, the plants
considered to be partially cold-acclimated and defined as
were allowed to adjust to set chamber conditions for 12 h.
autumn pine. For further cold acclimation, autumn pine
During experiments, the relative humidity of the air was
was transferred to a temperature regime of 5/5 C (day/
maintained at 50 and 75% for wheat and pine, respectively.
night), 75% humidity, a PFD of 250 mol m2 s1 (5 C/250
Whole-plant NCER of pine was measured under the tem-
PFD) and a maintained 8 h photoperiod. After a further
perature regimes of 25/15 C (day/night), 15/10 C and
period of 6 weeks these seedlings were defined as winter
5/5 C for summer, autumn and winter pine, respectively.
pine. Fully developed second-year needles of summer,
To elucidate the effect of short-term temperature changes
autumn and winter pine were used for CO2 gas exchange
on whole plant NCER, summer pine was cold stressed by
measurements, pigment analysis, gel electrophoresis and
shifting the plants to 5/5 C (25/15 C5/5 C), and both
fluorescence measurements. All data presented in this
autumn- and winter-acclimated pine were shifted to the 25/
paper are averages of three independent experiments with
15 C (15/10 C25/15 C and 5/5 C25/15 C) temperature
four replications for each experiment SE.
regime.
During measurements of whole-plant NCER in wheat,
Whole-plant net CO2 exchange measurements
the day/night temperature was maintained at 20/16 C for
The whole-plant net CO2 exchange analysis system has control plants and at 5/5 C for cold-acclimated plants.
been described in detail previously (Leonardos, Tsujita & Short-term temperature effects on NCER were assessed
Grodzinski 1994; Jiao, Leonardos & Grodzinski 1996). It after shifting control plants grown at 20/16 C to 5/5 C (20/

2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771

16 C5/5 C). Measurements under these temperature
regimes for wheat and pine were carried out under (1)
ambient air conditions [p(O2) 21 kPa and p(CO2) 35 Pa]
and 250 PFD, (2) ambient air and 1000 PFD, and (3) air
with enhanced CO2 concentration [p(CO2) 100 Pa] and
1000 PFD. During the measurements, each plant-holding
chamber contained either five pine seedlings or five pots
with wheat plants (10 plants per pot). In each experiment,
the total data were provided by series of measurements on
a minimum of four replicates (i.e. plantchamber combina-
tions). The leaf area measurements were obtained destruc-
tively at the end of the daily CO2 exchange measurements.
For technical reasons, the lowest measuring temperature
possible in the light was 75 2 C.
Chlorophyll a fluorescence measurements
All chlorophyll (Chl) a fluorescence measurements were
made using a PAM modulated fluorescence system (Heinz
Walz, Effeltrich, Germany) as described in detail by Savitch
et al. (2000b). The temperature of the chamber was con-
trolled to maintain a sample temperature of either 20 or
5 C for wheat plants and either 25, 15 or 5 C for pine
plants. All measurements were made 4 h after the onset of
the photoperiod. Light response curves of the quantum
yield of PSII electron transport (PSII), the photochemical
efficiency of open PSII reaction centres in light (Fv/Fm),
and the non-photochemical quenching of absorbed light
(NPQ) were measured under ambient air conditions. Fo and
Fm were determined for leaf segments dark adapted for 1 h.
Fo and Fm were determined after 2030 min when steady-
state rates of photosynthesis were attained. All fluores-
cence parameters were calculated according to Schreiber,
Bilger & Neubauer (1994).
Pigment analyses
Pigments were extracted from leaf samples by homogeni-
zation in 100% high-pressure liquid chromatography
(HPLC)-grade acetone with 03 mg L1 CaCO3 at 4 C in
dim light, and separated and analysed by HPLC (Gray et al.
1996). The retention times and response factors of -
carotene, lutein, Chl a and Chl b were determined by the
injection of known quantities of pure standards (Sigma,
Stockholm, Sweden), and the retention times and response
factors of the xanthophyll cycle pigments were determined
using pigments isolated from barley by thin layer chroma-
tography (TLC) (Hurry et al. 1992). To determine the Chl
a/b ratio, pigments were extracted in 80% acetone buffered
with 25 mM Hepes (pH 75) and quantified according to the
equations of Porra, Thompson & Kriedemann (1989). The
contents of pigment were expressed in g g1 fresh weight.
The epoxidation state (EPS) of the xanthophyll cycle pig-
ment pool was calculated as: EPS = (V + 05 A)/(V + A +
Z), where V, A, and Z correspond to the concentrations of
violaxanthin, antheraxanthin, and zeaxanthin, respectively.
Leaf samples for pigment analysis were for wheat plants
collected 1 h (morning) and 15 h (afternoon) after the onset
2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771
Light utilization and cold acclimation 763
of the photoperiod. Needles from summer pine were col-
lected 1 h (dark) before, and 1 h (morning) and 16 h (after-
noon) after the onset of the photoperiod. Needles from
autumn and winter pine were collected 1 h (dark) before,
and 1 h (morning) and 7 h (afternoon) after the onset of the
photoperiod.
Thylakoid preparation, sodium dodecyl
sulphate-polyacrylamide gel electrophoresis
and immunoblotting
Chloroplast thylakoids were isolated from pine needles
using an ice-cold buffer containing 50 mM Hepes (pH 76),
04 M sucrose, 10 mM MgCl2, and 20% (w/v) polyethylene
glycol 4000, and from wheat leaves using an ice-cold buffer
containing 50 mM Tricine (pH 78), 04 M sorbitol, 5 mM
MgCl2, and 10 mM NaCl. Plant material was homogenized
in a rotor for 3 min, filtered through miracloth and centri-
fuged at 9000 g for 20 min at 4 C. The pellet containing
chloroplasts was resuspended in a wash buffer containing
50 mM Tricine (pH 78), 5 mM MgCl2, and 10 mM NaCl.
After further centrifugation the thylakoid fractions were
resuspended in a buffer containing 01 M sorbitol in the
wash buffer.
In preparation for sodium dodecyl sulphate-polyacryla-
mide gel electrophoresis (SDS-PAGE) the chloroplast
thylakoid fractions were solubilized in a buffer containing
60 mM Tris-HCl (pH 78), 12% (w/v) sucrose, 2% (w/v) SDS
(SDS : Chl, 10 : 1), 1 mM ethylenediamine tetraacetic acid
disodium salt, and 58 mM dithiothreitol. Samples were
loaded for SDS-PAGE on an equal Chl basis (10 g Chl per
slot). Solubilized membrane polypeptides were separated
by SDS-PAGE using the buffer system of Laemmli (1970)
in a Mini-Protein II apparatus (Bio-Rad, Sundbyberg,
Sweden) as described in detail by Gray et al. (1996) with a
4% (w/v) stacking gel and a 12% (w/v) resolving gel. For
immunodetection, separated polypeptides were electro-
phoretically transferred (Mini-Trans Blot; Bio-Rad) to
nitrocellulose membranes (02 m pore size; Bio-Rad) as
described by Gray et al. (1996), and probed with monospe-
cific antibodies against D1, PsbS, Lhca4, Lhcb1, Lhcb5 and
PsaD. Blots were developed using goat antirabbit IgG con-
jugated with horseradish peroxidase (Sigma) as a second-
ary antibody. The complexes were visualized using a
chemoluminescent detection system (ECL Detection Kit;
Amersham, Uppsala, Sweden).
RESULTS
Daily carbon exchange
The effects of cold acclimation on whole plant net carbon
exchange rates (NCER) are shown for Lodgepole pine
(Fig. 1A, summer pine; C, autumn pine; E, winter pine)
and winter wheat (Fig. 1G; non-acclimated wheat; I, cold-
acclimated wheat). Notice that plants cold-acclimated
under a day/night temperature regime of 5/5 C were for
technical reasons measured at 75 2 C in the light (Fig. 1E
764 L. V. Savitch et al.

PINE
A. 25/15C B. 255C C. 15/10C D. 1525C E. 5/5C F. 525C

20

15

10

5
Whole plant NCER (mmol CO2 m2 s1)

0 17 24 41 48 0 8 24 32 48 0 8 24 32 48

Time (h)

WHEAT
G. 20/16C H. 205C I. 5/5C

15

10

0 16 24 40 48 0 16 24

Time (h)

Figure 1. Effects of cold acclimation on whole plant net carbon exchange rates (NCER) measured as CO 2 exchange in Lodgepole pine and
winter wheat. NCER were monitored first for 24 h at the growth temperatures of 25/15 C (day/night), 15/10 C and 5/5 C for summer,
autumn and winter pine, respectively (PINE: A, C, E). To elucidate the effect of short-term temperature changes on whole plant NCER, the
measurements continued over a second 24 h period with summer pine (25/15 C) being shifted to 5 C (255 C; PINE: B), and both autumn
pine (15/10 C) and winter pine (5/5 C) being shifted to the higher temperature regime of 25/15 C (1525 C and 525 C; PINE: D and
F). During the measurements, the 17 h photoperiod was maintained for the 25/15 C-grown pine and the 8 h photoperiod was maintained
for 15/10 C and 5/5 C-grown pine. Similarly, NCER in winter wheat was monitored for 24 h at the growth temperature regimes of 20/
16 C (day/night) and 5/5 C (WHEAT: A and C). The effect of short-term temperature changes on whole plant NCER was elucidated during
a second 24 h period with 20/16 C wheat being cold stressed at 5 C (205 C; WHEAT: B). During the measurements, the 16 h photoperiod
was maintained for all treatments. NCER measurements for pine and wheat were performed at the growth photon flux density of 250 mol
m2 s1 and a CO2 partial pressure of 35 Pa (open circles), at a high photon flux density of 1000 mol m2 s1 and a CO2 partial pressure of
35 Pa (filled circles), and at 1000 mol m2 s1 and an elevated CO2 partial pressure of 100 Pa (open triangles). All data are averages of three
independent experiments with four replications for each experiment. Data points were collected with an interval of 6 min.

& I). When comparing the effects of cold acclimation on
NCER under ambient growth light, air and temperature
conditions, pine responded with progressive inhibition. This
inhibition became even more pronounced when measure-
ments were done under high light (1000 mol m2 s1), or
when the high light was combined with elevated CO2 (100
Pa). This shows that the cold acclimation induced inhibition
of NCER in pine was not primarily controlled at the sto-
matal level, which is supported by earlier observations
(Strand & quist 1985). The inhibition of NCER by cold
acclimation (Fig. 1E) rather results from developmental
changes at the metabolic level, since just shifting 25/15
grown summer pine plants to a measuring temperature of
5/5 C (Fig. 1B) caused much less inhibition of NCER dur-
ing the day than observed after cold acclimation (Fig. 1E).
Likewise, shifting partially (autumn pine) and fully (win-
ter pine) cold-acclimated pine plants to a temperature
regime of 25/15 C did not increase NCER (Fig. 1D & F). In
fact, shifting to the higher temperature regime inhibited
NCER somewhat, particularly in the partially cold-accli-
mated autumn seedlings of pine (Fig. 1D).
In contrast to pine, wheat did not respond to cold accli-
mation with any inhibition of NCER (Fig. 1G & I). The par-
tial inhibition of NCER observed initially after shifting the

2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771
 Light utilization and cold acclimation 765

Table 1. Effects of day-time photon flux density (PFD) and p(CO2) on night respiration of warm grown, low-temperature stressed and cold
acclimated Lodgepole pine and winter wheat. The rates of whole-plant dark respiration (Rd, mol m2 s1) were measured at growth night
temperatures and were calculated by averaging NCER over the dark period: 7 h for 25 C/15 C and 25 C/15 C5 C/5 C pine; 16 h for
15 C/10 C and 5 C/5 C pine; 8 h for 20 C/16 C, 20 C/16 C5 C/5 C and 5 C/5 C wheat. Measurements of whole-plant NCER for
each environmental challenge were made every 6 min. Data represent the means SE for n = 70160

Variant 250 PFD, 35 Pa CO2 1000 PFD, 35 Pa CO2 1000 PFD, 100 Pa CO2

Pine
25 C/15 C (summer pine) 048 004 219 006 235 006
25 C/15 C5 C/5 C 113 005 177 006 170 009
15 C/10 C (autumn pine) 123 003 252 004 210 004
5 C/5 C (winter pine) 048 001 090 002 081 002
Wheat
20 C/16 C 101 002 143 001 098 002
20 C/16 C5 C/5 C 078 001 100 002 100 001
5 C/5 C 161 002 168 001 267 003

seedlings from a temperature regime of 20/16 C to 5/5 C
(Fig. 1H), was fully recovered in plants developed at 5/5 C
(Fig. 1I). In fact, measuring NCER under high light with or
without elevated CO2, showed that cold acclimation of
wheat increased the potential capacity for photosynthesis,
which is in sharp contrast to the inhibition observed in cold-
acclimated pine.
A comparison of NCER during the course of the light
period, also revealed a progressive decline of NCER in
cold-acclimated pine, particularly when measured under
high light and elevated CO2 conditions (Fig. 1E), whereas
the NCER of cold-acclimated wheat stayed largely constant
during the whole light period (Fig. 1I). This suggests feed-
back inhibition of photosynthesis due to a reduced sink
capacity for assimilates in dormant and cold-acclimated
pine. In contrast, the active growth and development of
cold-acclimated wheat is likely to sustain a high demand for
assimilates even after cold acclimation thus preventing feed
back inhibition of photosynthesis at low temperatures.
In quantitative terms, the effects of cold acclimation on
the rate of whole plant dark respiration were minor under
more, the carbon gained during the light period was largely
respired during the long night period of 16 h. This resulted
in the net carbon gain over a 24 h light/dark cycle for cold-
acclimated autumn pine and winter pine being only
about 20% of that observed in the control summer pine
grown at 25/15 C. Notice that the full effect by cold accli-
mation on NCER was largely reached already during the
first phase of cold acclimation combining short day (8 h)
with a moderate temperature lowering (15/10 C) to termi-
nate growth and induce dormancy (Bigras et al. 2001). In
contrast, the daily carbon gain of wheat was unaffected by
cold acclimation (Fig. 2B). This strong retardation of the
daily carbon gain in cold-acclimated pine correlates very
PINE WHEAT
6 A B
Carbon gain (g C m2)

ambient growth light, air and temperature conditions for

both pine and wheat (Table 1). However, the rate of dark
4
respiration increased 60% in wheat after cold acclimation
(250 PFD, 35 Pa CO2), whereas in pine the effect of cold
acclimation on dark respiration was negligible with
increased dark respiration seen only during the course of 2
cold acclimation at a day/night temperature regime of
15/10 C (250 PFD, 35 Pa CO2). When increasing the light
to a PFD of 1000 mol m2 s1, and when combining high 0
light and elevated CO2, respiration increased further in
both pine and wheat with the strongest increases in non- 0 24 0 24
acclimated and partially cold-acclimated pine and in cold-
acclimated wheat. Time (h)
The daily carbon gain under ambient light, air and tem-
perature conditions was calculated from the NCER data of Figure 2. Effects of cold acclimation on the carbon gain of
Fig. 1. In pine, the effects on NCER by cold acclimation Lodgepole pine and winter wheat. Presented data reflect the
carbon gain of (A) 25/15 C (solid line), 15/10 C (dashed line) and
resulted in a strong retardation of the carbon gain during
5/5 C (dotted line)-grown Lodgepole pine, and (B) 20/16 C (solid
the light period (Fig. 2A). This was due both to the short- line) and 5/5 C (dotted line)-grown winter wheat over a 24 h
ened photoperiod from 16 to 8 h, and to the reduced rate of period. The growth photon flux density of 250 mol m2 s1 and a
photosynthesis (reduced slope of carbon gain). Further- CO2 partial pressure of 35 Pa was used.

2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771
766 L. V. Savitch et al.

The yield of photochemistry

PINE WHEAT
The photochemical efficiency of open PSII reaction centres
(Fv/Fm), and the photochemical yield of PSII electron
08 A B
transport (PSII) as derived from fluorescence kinetics
measurements under ambient air conditions were plotted
06
F 'v /F 'm

as a function of irradiance for non-acclimated and cold-

04 acclimated leaves of Lodgepole pine and winter wheat (Fig.
3A, B, E & F). In accordance with the gas exchange mea-
02 surements (Fig. 1E), cold acclimation of pine resulted in
strong reductions of both the efficiencies of open PSII reac-
tion centres and the photochemical yields of PSII electron
20 C D transport at all PFDs studied, whereas the effects by cold
acclimation on PSII photochemistry in wheat were mini-
15
NPQ

mal. The low efficiency or yield of PSII photochemistry in

10
pine occurred together with a partial loss of PSII reaction
centres as indicated by decreased levels of the reaction cen-
05 tre D1 protein (Fig. 4A). There was no apparent loss of D1
protein in cold-acclimated wheat (Fig. 4B). Clearly, the
decreased rates of photosynthesis observed for cold-
acclimated pine correlated well with both a decreased yield
08 E F
of PSII photochemistry and a partial loss of PSII reaction
centres. In contrast to PSII, PSI of Lodgpole pine did not
06
respond to cold acclimation by a decrease as indicated by
FPSII

04 the marker PsaD protein that did not change in neither pine
or wheat. This is in agreement with earlier findings that win-
02 ter stress has very little effect of PSI photochemistry in
Scots pine (Ivanov et al. 2001)

300 600 900 300 600 900

2 1
Light irradiance (mmol m s )
Figure 3. Effects of cold acclimation on Chl fluorescence
parameters in needles and leaves of Lodgepole pine (A, C, E) and
winter wheat (B, D, F). The light response curves of Fv/Fm, NPQ
and PSII were measured at a p(CO2) of 35 Pa and the growth
temperatures of 25 C and 20 C for 25/15 C pine and 20/16 C
wheat, respectively (open circles), and at 5 C for 5/5 C cold-
acclimated pine and wheat (filled circles). Data represent mean
SE for n = 5.
well with the fact that pine enters dormancy during cold
acclimation, whereas the high rate of carbon gain during
cold acclimation in wheat correlates with continued growth
and development during cold acclimation.
Changes in pigmentation and non-
photochemical dissipation of absorbed light
Cold acclimation reduced the Chl content of Lodgepole
pine needles, but had no significant effect on the Chl con-
tent of winter wheat leaves, with only minor changes in the
Chl a/b ratio in both species (Table 2). The Chl loss in pine
was already detectable during the early phase of cold accli-
mation when a short day (8 h), and a moderate temperature
decrease (15/10 C), were combined to terminate primary
growth and to induce dormancy. We also analysed the
amount of light-harvesting Chl protein complexes. PSI and
PSII have specific light-harvesting Chl proteins that consti-
tute the inner antennae. Most Chl, however, is associated
with the major, trimeric LHCII complex that associates to
both PSI and PSII. We chose three proteins, Lhca4, Lhcb5

Table 2. Effects of cold acclimation on the total Chl (a + b) content (g g1 fresh weight), the Chl a/b ratio (w/w) and the carotenoid/Chl
(a + b) ratio (w/w) in needles and leaves of Lodgepole pine and winter wheat, respectively. The carotenoid/Chl (a + b) ratios were calculated
from primary data on Chl content and total carotenoids (Tables 3 & 4). Data represent the means SE for n = 4

Pine Wheat

25 C/15 C 15 C/10 C 5 C/5 C 20 C/16 C 5 C/5 C

Parameters summer pine autumn pine winter pine

Chl (a + b) 2182 148 1342 81 1497 66 2113 137 2155 71

Chl a/b 32 02 33 01 30 01 32 01 31 01
Carotenoid/Chl (a + b) 021 030 031 015 019

2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771
 Light utilization and cold acclimation 767

Figure 4. Western blots of SDS-PAGE probed

with antibodies raised against: the PSII reaction
centre D1 protein, the light-harvesting Chl binding
proteins Lhcb1, Lhca4 and Lhcb5, the Chl binding
protein PsbS, and the PSI ferredoxin binding PsaD
protein of thylakoids isolated from (A) Lodgepole
pine acclimated to the day/night temperature
regimes of 25/15 C, 15/10 C and 5/5 C, and from
(B) winter wheat acclimated to the day/night
temperature regimes of 20/16 C and 5/5 C, with
the day temperatures being used to mark the
different PAGE lanes. Samples were collected at
indicated growth temperatures and at the growth
photon flux density fo 250 mol m2 s1. Lanes of
SDS-PAGE were loaded on an equal Chl basis.
Numbers on the right represent molecular (kDa)
masses of markers.

and Lhcb1 as representatives of the inner antenna of the
two photosystems and trimeric LHCII, respectively (Jans-
son 1994). Figure 4A shows that the inner antenna of PSI
was not affected by cold acclimation in Lodgepole pine, the
inner antenna of PSII decreased to a similar extent as the
reaction centre D1 protein, whereas the trimeric LHCII
had decreased more significantly already during the early
phase of cold acclimation. This is in line with the observa-
tion by Vogg et al. (1998), showing a major Chl loss in Scots
pine after short day treatment to induce dormancy, which
constitutes an important developmental phase for the
induction of maximum freezing tolerance during cold accli-
mation of conifers (Levitt 1980). In contrast, cold acclima-
tion induced no major changes in the content of Lhcb1 in
wheat and the inner antennae Lhcb5 and Lhca4 were also
unaffected (Fig. 4B).
Cold acclimation induced increases in lutein content in
both pine and wheat, a decrease in -carotene in pine, and
a moderate increase of the xanthophyll cycle pigments (vio-
laxanthin + antheraxanthin + zeaxanthin) in both pine and
wheat (Tables 3 & 4). The observed Chl and carotenoid
changes resulted in increased carotenoid/chlorophyll ratios

Table 3. Effect of cold acclimation on the content of carotenoids (g g1 fresh weight) and the epoxidation state (EPS) of the xanthophylls
pool in needles of Lodgepole pine . Samples were collected at prevailing growth conditions (see Material and Methods for details). EPS was
based on primary data on violaxanthin (V), anteraxanthin (A) and zeaxanthin (Z). Data represent the means SE for n = 4

25 C/15 C(summer pine) 15 C/10 C (autumn pine) 5 C/5 C (winter pine)

Parameters Morning Afternoon Dark Morning Afternoon Dark Morning Afternoon Dark

Lutein 114 8 190 5 144 1 146 11 144 6 247 6 155 10 244 4 171 1
-Carotene 67 3 101 3 91 1 19 2 23 1 77 1 21 1 30 1 20 1
-Carotene 78 2 121 6 60 1 85 5 72 2 57 1 72 4 110 3 77 1
Neoxanthin 58 2 66 2 61 2 42 1 39 1 48 1 42 2 72 2 43 1
Violaxanthin 68 2 65 4 73 1 54 2 34 1 80 1 33 1 58 1 57 1
Anteraxanthin 23 04 6 7 03 62 00 48 03 173 02 77 13 295 09 366 18 155 08
Zeaxanthin 0 74 04 0 0 128 23 0 244 14 380 61 108 15
V+A+Z 70 2 79 3 79 1 59 3 64 3 88 2 91 5 132 7 89 3
EPS 098 092 096 096 066 096 058 057 073

2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771
768 L. V. Savitch et al.

Table 4. Effect of cold acclimation on the content of carotenoids NPQ value for cold-acclimated wheat at low, limiting PFDs
(g g1 fresh weight) and the epoxidation state (EPS) of the was only about 25% of its maximum measured at high light.
xanthophylls pool in leaves of winter wheat. Samples were Thus, the absolute value for NPQ for cold-acclimated
collected at prevailing growth conditions (see Material and
wheat at low PFDs was about 50% less than observed for
Methods for details). EPS was based on primary data on
violaxanthin (V), anteraxanthin (A) and zeaxanthin (Z). Data pine at the same PFD (Fig. 3C & D).
represent the means SE for n = 4. Using mutants of Arabidopsis, Li et al. (2000) have
shown that the chlorophyll binding PsbS protein (CP22)
20 C/16 C 5 C/5 C described by Funk et al. (1995) is essential for the zeaxan-
thin-dependent excitation energy quenching. Circumstan-
Parameters Morning Afternoon Morning Afternoon tial evidence also indicates that PsbS is the actual site for
Lutein 107 14 177 16 159 25 171 12 the quenching (Andersson et al. 2001). In line with this find-
-Carotene 99 11 133 13 119 15 58 10 ing, the lower EPS ratio and the increased level of sustained
Neoxanthin 35 1 60 5 66 4 36 2 non-photochemical quenching observed in cold-acclimated
Violaxanthin 34 4 52 5 57 3 41 2 autumn and winter pine correlated very well with a strong
Anteraxanthin 32 08 20 04 18 04 06 02
induction of the PsbS protein (Fig. 4A), whereas the level of
Zeaxanthin 0 0 0 0
V+A+Z 63 6 54 5 58 3 42 1 PsbS was unchanged in cold-acclimated winter wheat as
EPS 092 097 095 099 was EPS, and NPQ was relatively less effected by cold accli-
mation in wheat than in pine.

(Table 2), particularly for pine. In addition, the de-expoxi-

dation of violaxanthin to antheraxanthin and zeaxanthin
increased strongly in the pine needles during cold acclima-
tion as reflected by decreasing values of the epoxidation
state with a sustained level of de-epoxidation by the end of
the dark period in winter pine acclimated to a temperature
regime of 5/5 C (EPS; Table 3). However, cold acclimation
of wheat leaves caused no de-epoxidation of violaxanthin
as shown by consistently high EPS values for both cold-
acclimated and non-acclimated wheat leaves (EPS; Table
4). Increased carotenoid/chlorophyll ratios, and low EPS
ratios for the xanthophyll cycle pigments, have been corre-
lated to increased capacities for non-photochemical dissi-
pation of light absorbed by the photosynthetic antennae
(Demmig-Adams & Adams 1992; Niyogi 1999; Mller, Li &
Niyogi 2001).
Apparently, pine and wheat show totally different pig-
ment responses during cold acclimation: pine not only
reduces its Chl absorbing capacity by decreasing the needle
Chl content and the number of PSII centres, it also invests
in an increased capacity for sustained non-photochemical
dissipation of excited Chl. In contrast, wheat maintains its
light-absorbing capacity with minimal changes in its sus-
tained capacity for non-photochemical dissipation of
excited Chl. Accordingly, the carotenoid changes observed
as a result of cold acclimation were corroborated by mea-
surements of the capacity for non-photochemical quench-
ing (NPQ) of absorbed light, as derived from fluorescence
kinetics measurements and expressed as a function of irra-
diance (Fig. 3C & D). The sustained capacity for NPQ was
about three-fold higher for cold-acclimated pine at low, lim-
iting PFDs than observed for non-acclimated pine (Fig.
3C). As expected, as the measuring PFD increased, NPQ
also increased for both cold-acclimated and warm-accli-
mated pine needles. Clearly, even at low, limiting PFDs,
cold-acclimated pine was already at 75% of its maximum
NPQ observed at high light. Although cold acclimation did
enhance NPQ in wheat at all light levels measured, the
DISCUSSION
It is well established that conifers and winter cereals have
somewhat different requirements for cold acclimation
(Levitt 1980). Conifers such as Lodgepole pine require a
shortened day length for the induction of terminal buds and
for the cessation of associated, primary growth. In combi-
nation with exposure to chilling temperatures, this charac-
teristic autumnal response in development also results in
the freezing resistance required for overwintering even
under harsh, sub-Arctic winter conditions with prevailing
temperatures well below zero (Bannister & Neuner 2001).
In contrast, winter cereals such as wheat do not respond to
a shortened photoperiod with the termination of growth.
On the contrary, winter cereals continue to grow and estab-
lish themselves throughout the autumn. Active growth and
development at chilling autumn temperatures with occa-
sional frost nights is in fact a requirement for the induction
of maximum freezing tolerance (Fowler & Carles 1979;
Krol, Griffith & Huner 1984; Griffith & McIntyre 1993).
Both cold temperate conifers and winter cereals are among
the most frost-resistant plants with retained foliage during
autumn and winter (Levitt 1980).
In the context of these different growth strategies for
conifers and winter cereals, we understand that the require-
ment for photosynthetic assimilates also differ, with a
decreased requirement for pine and the maintenance of a
high demand for winter cereals after cold acclimation. In
concert with these different demands for photosynthetic
assimilates during the autumn, we show that Lodgepole
pine decreases both its capacity (Fig. 1E) and efficiency
(Fig. 3A & E) of photosynthesis after cold acclimation,
whereas winter wheat maintains both its capacity (Fig. 1I)
and efficiency (Fig. 3B & F) of photosynthesis after cold
acclimation. The mechanisms behind the relationship
between photosynthesis and growth is not known, but feed
back and feed forward mechanisms are certainly involved
(Labate & Leegood 1988; Sharkey 1990; Savitch, Harney &

2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771

Huner 2000a). The limited sink capacity for photosynthetic
assimilates due to the retarded growth of pine after cold
acclimation is likely to result in feedback inhibition
decreasing the rate of CO2 uptake during the course of the
day. This assumption is supported by the observation of a
gradual suppression of CO2 uptake during the course of the
light period in cold-acclimated pine, particularly under high
light and elevated CO2 conditions (Fig. 1C & E). In con-
trast, winter wheat with a high sink capacity due to active
growth and development showed no signs of feedback sup-
pression of the rate of photosynthesis during cold acclima-
tion, at least not under ambient air conditions. This is
further supported by earlier observations that cold acclima-
tion of winter cereals strongly increase the capacity for cel-
lular sugar metabolism (Hurry et al. 1995), and the storage
of assimilates as fructans (Savitch et al. 2000a), thus adding
to a high sink capacity for assimilates.
The down-regulation of photosynthesis in cold-accli-
mated pine was reflected in partial losses of PSII reaction
centres and associated light-harvesting Chl antennae,
whereas PSI was unaffected (Fig. 4A; Table 2). This
response clearly reduces light absorption and photochem-
istry per needle area, and helps to adjust photosynthesis to
match the diminishing demand for assimilates. In addition,
the de-epoxidation of the xanthophyll-cycle pigments to
zeaxanthin resulting in low and partially sustained values of
EPS (Table 3), provide efficient means to dissipate light
energy absorbed in excess to what can be orderly dissipated
through photochemistry. The exact mechanism of zeaxan-
thin quenching of the excitation energy absorbed by the
Chl antennae has not yet been worked out, but it has
recently been shown that the PsbS pigment-binding protein
is essential for the process (Li et al. 2000). In accordance
with this, we also observe a strong induction of the PsbS
protein during the course of cold acclimation of pine (Fig.
4A). It is important to notice that in Lodgepole pine it is the
autumn acclimation phase, combining a shortened photo-
period with a moderate lowering of the temperature (from
25/15 C to 15/10 C), which has the strongest effects on the
Chl reduction, on the increase of the carotenoid/chloro-
phyll ratio (Table 2), and on the induction of the PsbS pro-
tein (Fig. 4A). Termination of growth and the induction of
dormancy as induced by short days apparently also prepare
for the winter by reducing the light-harvesting capacity, and
by down-regulating photochemistry, both by reducing the
number of PSII reaction centres and by increasing the non-
photochemical quenching capability.
In a natural stand of Scots pine (Pinus sylvestris L.) in
Northern Sweden, photosynthesis becomes almost com-
pletely downregulated during the winter months (Leverenz
& quist 1987). Our results for Lodgepole pine under con-
trolled growth conditions are consistent with the depres-
sion of photosynthesis under natural winter conditions
which is associated with a partial loss of Chl, an almost com-
plete loss of PSII reaction centres, a low EPS value due to
a sustained de-epoxidation of xanthophylls to antheraxan-
thin and zeaxanthin, and an accumulation of the PsbS pig-
ment-binding protein as part of aggregated light-harvesting
2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 761771
Light utilization and cold acclimation 769
Chl protein complexes (Ottander et al. 1995). We view this
autumn- and winter-induced down-regulation of the func-
tion of photosynthesis, characterized by a high capacity for
non-photochemical dissipation of excited Chl, as an impor-
tant acclimative response to protect from irreparable
photo-oxidative damage of the needle foliage when prevail-
ing freezing winter temperatures prevent normal photosyn-
thesis. As conifers also have the ability to recover fully from
this down-regulated state during the spring (Ottander &
quist 1991; Ottander et al. 1995), this capacity to down-
regulate photosynthesis is an important mechanism for the
successful establishment of evergreen conifers in cold tem-
perate and sub-Arctic climates. The evergreen winter strat-
egy provides a means for capitalizing on made investments
in assimilating area, and it may be of particular importance
in environments with poor nutritional conditions (Schulze
1982).
In contrast to Lodgepole pine, winter wheat is not dor-
mant during cold acclimation but requires growth and
development at low temperatures to exhibit maximum
freezing tolerance (Fowler & Carles 1979; Krol et al. 1984;
Griffith & MacIntyre 1993; Pocock et al. 2001). This is
reflected in the maintenance of a high capacity for photo-
synthesis in cold-acclimated leaves (Fig. 1I), the mainte-
nance of a high light-absorbing capacity per unit leaf area
(Table 2), and a minimal investment in the capacity for non-
photochemical dissipation of excited Chl (Fig. 4B; Table 4).
Clearly, winter wheat maintains photosynthesis as the
major quencher of excited Chl even in the cold-acclimated
state, which is consistent with a high demand for assimilates
to sustain active growth and development throughout the
autumn months. Adams and coworkers (Verhoeven,
Adams & Demmig-Adams 1998, 1999; Adams et al. 2001)
also relate a high potential rate of winter photosynthesis in
mesophytes to a disengagement of xanthophyll quenching,
whereas a suppressed rate of winter photosynthesis in
schlerophyllous evergreens correlates with a sustained
down-regulation of photosynthesis through activated xan-
thophyll quenching.
In conclusion, we view the strategy of pine to partially
reduce its Chl antenna and invest in a sustained high, non-
photochemical energy-dissipating capacity during cold
acclimation, as an adjustment to avoid photo-oxidative
damage and leaf death during the winter when the normal
dissipation of excitation energy through photosynthesis is
largely prevented. Without this acclimative strategy during
the induction of dormancy, it is unlikely that evergreen
conifers would have managed to successfully adapt to cold
temperate and sub-Arctic climates. Winter cereals, do on
the other hand not require a sustained high capacity for
non-photochemical dissipation of absorbed light since, as
the seedlings have established themselves during the
autumn, they normally become covered by snow and it is
the crown of the seedlings that survive the winter months
rather than the leaves that developed and showed high
activities of photosynthesis during autumn. During the
autumn, photo-assimilates are translocated to the crown
(Savitch et al. 2000a) which provides the necessary energy
770 L. V. Savitch et al.
and carbon pool for the new growth in the following
spring.
ACKNOWLEDGMENTS
This work was supported by grants from the Swedish Foun-
dation for International Cooperation in Research and
Higher Education (G. and N.P.A.H.), the Swedish
Research Council (G..), the Swedish Forestry and Agri-
cultural Research Council (S.J.), and the Natural Sciences
and Engineering Research Council of Canada (N.P.A.H.).
The research was also supported by grants to B.G. from the
Natural Sciences and Engineering Research Council of
Canada, the Center for Research in Earth and Space Tech-
nology, the Ontario Ministry of Agriculture and Food and
Rural Affairs, Flowers Canada Inc., and the Canadian
Adaptation Council.
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Received 15 August 2001; received in revised form 16 January 2002;
accepted for publication 1 February 2002