Plant, Cell and Environment (2009) 32, 859–874
doi: 10.1111/j.1365-3040.2009.01965.x
Adjustment of growth, starch turnover, protein content and
central metabolism to a decrease of the carbon supply
when Arabidopsis is grown in very short photoperiods
YVES GIBON1,2, EVA-THERESA PYL1, RONAN SULPICE1, JOHN E. LUNN1, MELANIE HÖHNE1,
MANUELA GÜNTHER1 & MARK STITT1
1
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Golm , Germany and 2INRA Bordeaux,
University of Bordeaux 1&2, UMR619 Fruit Biology, F-33883 Villenave d’Ornon, France
ABSTRACT
Arabidopsis was grown in a 12, 8, 4 or 3 h photoperiod
to investigate how metabolism and growth adjust to a
decreased carbon supply. There was a progressive increase
in the rate of starch synthesis, decrease in the rate of starch
degradation, decrease of malate and fumarate, decrease of
the protein content and decrease of the relative growth rate.
Carbohydrate and amino acids levels at the end of the night
did not change. Activities of enzymes involved in photosynthesis, starch and sucrose synthesis and inorganic nitrogen
assimilation remained high, whereas five of eight enzymes
from glycolysis and organic acid metabolism showed a significant decrease of activity on a protein basis. Glutamate
dehydrogenase activity increased. In a 2 h photoperiod, the
total protein content and most enzyme activities decreased
strongly, starch synthesis was inhibited, and sugars and
amino acids levels rose at the end of the night and growth
was completely inhibited. The rate of starch degradation
correlated with the protein content and the relative growth
rate across all the photoperiod treatments. It is discussed
how a close coordination of starch turnover, the protein
content and growth allows Arabidopsis to avoid carbon
starvation, even in very short photoperiods.
Key-words: carbon metabolism; enzyme activities; fumarate; malate; starch.
INTRODUCTION
Plants are subjected to continual changes in the supply of
carbon (C) (Geiger, Servaites & Fuchs 2000; Paul & Foyer
2001; Smith & Stitt 2007; Stitt et al. 2007). One of the most
pervasive is the daily alternation between photosynthetic C
fixation in the light and respiration during the night. Most
plants buffer these diurnal changes by retaining part of the
photosynthate as starch in the leaves, and remobilizing it at
night to support respiration and continued export of C to
the rest of the plant (Geiger & Servaites 1994; Smith,
Denyer & Martin 1997; Geiger et al. 2000; Stitt et al. 2007).
Correspondence: M. Stitt. Fax: +00 49 331 567 8101; e-mail: stitt@
mpimp-golm.mpg.de
© 2009 Blackwell Publishing Ltd
Starch is typically degraded in a near-linear manner, with
5–10% remaining at the end of the night (see e.g. Fondy &
Geiger 1985; Geiger & Servaites 1994; Matt et al. 1998;
Gibon et al. 2004b; Smith et al. 2004). Slower changes in the
environment, which are superimposed on the diurnal cycle,
alter the amount of C fixed per 24 h cycle. Plants that are
grown in shorter light periods, lower light intensities or
lower CO2 concentrations synthesize proportionally more
starch in the light and degrade it more slowly at night (Stitt,
Bulpin & Ap Rees 1978; Chatterton & Silvius 1979, 1980,
1981; Mullen & Koller 1988; Lorenzen & Ewing 1992; Matt
et al. 2001; Gibon et al. 2004a).As a result, a small amount of
starch still remains at the end of the night.
The consequences of a transient imbalance between the
supply and utilization of C are dramatically illustrated in
mutants that are defective in the synthesis or degradation of
starch. Such mutants are able to grow in continuous light or
very long day regimes, but their growth is inhibited when
the night becomes longer (Caspar, Huber & Somerville
1985; Lin et al. 1988; Gibon et al. 2004b; Zeeman, Smith &
Smith 2007). In the starchless pgm mutant, sugars accumulate to high levels in the light but are rapidly depleted in the
first hours of the night (Caspar et al. 1985). This is followed
by an inhibition of growth, which is not reversed for several
hours in the following light period (Gibon et al. 2004b;
Bläsing et al. 2005; Stitt et al. 2007). Another example illustrating the importance of balancing the supply and utilization of C relates to seed abortion. Seed abortion in response
to a sudden episode of drought or heat stress is often a
consequence of C depletion rather than a direct effect of
these stresses on seed growth (Boyle, Boyer & Morgan
1991; Guilioni, Wery & Lecoeur 2003; McLaughlin & Boyer
2004a,b; Mäkelä, McLaughin & Boyer 2005).
The adjustment of starch turnover to a decreased C
supply will have to be accompanied by a decrease in the
rate of C utilization for respiration and growth (Smith &
Stitt 2007; Stitt et al. 2007). Changes of the C supply lead to
marked changes in the expression of thousands of genes
(Price et al. 2004; Li et al. 2006; Osuna et al. 2007). Many
C-regulated genes show marked changes of expression
during the night or the first hours of an extended night
(Thimm et al. 2004; Bläsing et al. 2005; Usadel et al. 2008).
859
860 Y. Gibon et al.
Modelling of global transcriptional responses confirmed
that these diurnal changes are caused by C signalling
(Usadel et al. 2008). They affect genes that encode enzymes
of primary metabolism and many genes that are required
for protein synthesis and cellular growth (Gibon et al.
2004b, 2006; Usadel et al. 2008). However, most enzyme
activities and the overall protein level do not show diurnal
changes (Gibon et al. 2004b). Further, although the large
diurnal changes of sugars in the starchless pgm mutant
result in accentuated diurnal changes of transcripts, the
encoded enzyme activities do not show larger diurnal
changes; instead, they shift towards the values found in
wild-type plants after several days of darkness (Gibon et al.
2004b, 2006). This led us to propose that diurnal changes of
transcripts are integrated over time as changes of enzyme
activities. The time delay between the changes of transcripts
and the encoded enzymes would allow information to be
extracted from the ‘noise’ of diurnal changes, and facilitate
gradual adaptation to sustained changes in the environment. This proposal implies that measurements of metabolic parameters like enzyme activities could provide a
useful tool to study how metabolism adjusts to changes in
the diurnal supply of C.
The following experiments were carried out to test the
capacity of Arabidopsis to grow in extremely short-day conditions, and to investigate the accompanying changes in
starch turnover, protein content and the activities of 25
enzymes from central metabolism. The results show that
Arabidopsis continues to grow in extremely short photoperiods, albeit at a strongly reduced rate. This is accompanied
by large and coordinated changes in C allocation, the
protein content and central metabolism.
MATERIAL AND METHODS
Plant growth
Arabidopsis thaliana var Col-0 was grown in soil [GS 90 soil
mixed with vermiculite in a ratio 2:1 (v/v)]. After germination the seedlings were grown for 1 week in a 16/8 h light
(250 mE m-2 s-1, 20 °C)/dark (6 °C) regime, for 1 week in an
8 h light (160 mE m-2 s-1, 20 °C)/16 h dark (16 °C) regime,
and then replanted with one seedling per pot and transferred for 1 week to growth cabinets with an 8/16 h light–
dark cycle (160 mE m-2 s-1, 20 °C throughout the day/night
cycle). The plants were then distributed into small growth
cabinets with a 12, 8, 4, 3 or 2 h photoperiod (all with a light
intensity of 160 mE m-2 s-1 and 20 °C throughout the day/
night cycle). The plants were harvested 17 d later. Typically,
at least five separate samples, each containing three
rosettes, were harvested.
Chemicals and enzymes
Inorganic compounds were purchased from Merck (Darmstadt, Germany), organic compounds from Sigma
(Taufkirchen, Germany), except ethanol (Merck) and
NADH (Roche), and enzymes from Roche (Mannheim,
Germany), except phosphoglycerate kinase (Sigma).
Glycerokinase is required for several of the enzyme assays.
As commercial sources of glycerokinase are not longer
available, yeast glycerokinase was overexpressed in
Escherichia coli as a His6-tagged fusion protein by cloning
the coding region of the Pichia farinosa GUT1 gene (kindly
provided by Prof. Cândida Lucas, Universidade do Minho,
Portugal) between the NcoI and EcoRI sites of expression
plasmid pETM11 (Günter Stier, EMBL, Heidelberg,
Germany). The protein was expressed in E. coli strain
Rosetta™ (Novagen, Darmstadt, Germany) carrying the
pLysS/RARE plasmid, and purified by immobilized metal
affinity chromatography on Talon™ (Co2+) metal affinity
resin (Clontech Laboratories Inc., Mountain View, CA,
USA) as described in Lunn et al. (2006) for the E. coli
trehalose-6-phosphate synthase. The purified glycerokinase
had a specific activity of 50 mmol min-1 mg-1protein, and
was stable for at least 6 months when stored at 4 °C as a
suspension in 80% (v/v) saturated (NH4)2SO4.
Extraction and assay of enzymes
In all cases, the entire sample was powdered under liquid
nitrogen and stored at -80 °C until its use. Aliquots of
~20 mg fresh weight (FW) were weighed out at -180 °C,
and were extracted by vigorous shaking with 1 mL of
extraction buffer, leading to an initial ~50-fold (w/v) dilution. The composition of the extraction buffer was 20%
(v/v) glycerol, 0.25% (w/v) bovine serum albumin, 1% (v/v)
Triton-X100, 50 mm HEPES/KOH pH 7.5, 10 mm MgCl2,
1 mm EDTA, 1 mm EGTA, 1 mm benzamidine, 1 mm
e-aminocaproic acid, 1 mm phenylmethylsulfonylfluoride,
10 mm leupeptin and 0.5 mm dithiothreitol. Phenylmethylsulfonylfluoride was added just prior to extraction. Enyzme
activities were assayed using a robotized platform as
described in Gibon et al. (2004b, 2006) and Sulpice et al.
(2007). The assay for cytosolic and plastidial phosphoglucose isomerases (cPGI and pPGI, respectively) was adapted
from Kruckeberg et al. (1989) by scaling down to the microplate format, and validated using A. thaliana RNAi lines
with no pPGI activity (unpublished results).
Extraction and assay of metabolites and
protein content
Sucrose, glucose, fructose were determined in ethanolic
extracts as in Jelitto et al. (1992); starch was determined as
in Hendriks et al. (2003); protein content as in Bradford
(1976); and malate and fumarate as in Nunes-Nesi et al.
(2007). Assays were prepared in 96 well microplates using a
Janus pipetting robot (Perkin-Elmer, Zaventem, Belgium).
The absorbances were read at 340, 570 or 595 nm in a
Synergy, an ELX-800 or an ELX-808 microplate reader
(Bio-Tek, Bad Friedrichshall, Germany).
Statistics
Standard procedures were carried out using functions of the
Excel program. Linear regression analysis was performed in
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
R software (R Development Core Team 2006). Hierarchical
clustering using Euclidian distance and average linkage
(Eisen et al. 1998) was performed on data expressed as log2
ratios to the respective controls (data from 12 h day/12 h
night samples) using the MultiExperiment Viewer software
(http://www.tm4.org/mev.html, Dana-Farber Cancer Institute, Boston, MA, USA).
(a)
0.12
Relative growth rate
(gFW·gFW−1·d−1)
Adaptation to short photoperiods 861
0.10
0.08
0.06
0.04
0.02
RESULTS
0
Plant growth
Levels of carbohydrates, amino acids and
organic acids
Short photoperiods led to lower levels of carbohydrates at
the end of the day (see Fig. 2). This was mainly caused by a
decrease of starch. The amount of starch synthesized during
the light period is equivalent to the amount degraded
during the night, and will be referred to as the diurnal starch
turnover. Diurnal starch turnover decreased from 31 mmol
hexose equivalents gFW-1 in a 12 h photoperiod to 27, 20
and 15 mmol hexose equivalents gFW-1 in 8, 4 and 3 h photoperiods, respectively (see Fig. 2). Diurnal starch turnover
was normalized on the length of the light and dark period to
calculate the rates of starch synthesis (mmol hexose equivalents incorporated into starch gFW-1 per hour light period)
and degradation (mmol hexose equivalents mobilized from
starch gFW-1 per hour dark period). To do this, we assumed
that synthesis and degradation occur linearly during the day
3
4
8
12
Photoperiod (h)
(b)
Average flux to/from starch
(mmol hexose·gFW−1·h−1)
After germination and seedling establishment in standard
growth conditions, 21-day-old plants were transferred to
a series of different photoperiod regimes, that is, 12 h
light/12 h dark, 8 h light/16 h dark, 4 h light/20 h dark, 3 h
light/21 h dark or 2 h light/22 h dark. Plant FW was approximately 0.12 g at the time of transfer. The plants were harvested 17 d later. Plant FW at harvest in the five regimes
was 0.83, 0.46, 0.26, 0.19 and 0.14 g, respectively. The
average relative growth rate (RGR; Fig. 1a) was calculated
assuming that growth rates were constant between the time
of transfer and harvest. This was checked for the 12 h photoperiod treatment in separate experiments (Tschoep et al.
2009). Growth decreased progressively as the light period
was shortened, but was still possible even in the 3 h light/
21 h dark photoperiod. There was effectively no growth in
the 2 h light/22 h dark regime (Fig. 1); by 40 d after transfer,
>50% of the plants were dead (data not shown).
Starch, sucrose, reducing sugars, total amino acids,
organic acids and total protein (Fig. 2) were measured at
the end of the day and at the end of the night. The results
will be presented first for photoperiods between 12 and 3 h,
and then for the extreme 2 h photoperiod. They will be
compared with the published data from experiments in
which Col-0 was transferred at the end of the night to
continuous darkness for 2, 3 and 7 d, and in which the
starchless pgm mutant was grown in a 12 h light/12 h dark
regime (Gibon et al. 2004b, 2006).
2
6
Degradation
5
Synthesis
4
3
2
1
0
2
3
4
8
12
Photoperiod (h)
Figure 1. Relative growth rates and starch turnover in different
photoperiods. Plants were grown for 3 weeks in standard
conditions, and then transferred to a 12/12, 8/16, 4/20, 3/21 or
2/22 h light/h dark photoregime. (a) Relative growth rates. These
were calculated from the rosette weight at the time of transfer
[120 mg fresh weight (FW)] and the weight after 17 d in the new
regime. The results are the mean ⫾ SE of five replicate samples.
(b) Average rates of starch synthesis and degradation. These
were estimated from the average levels of starch at the end of
the light period and the end of the night. The insert in panel
(a) shows the relation between the estimated relative growth rate
and the average rate of starch degradation during the night.
and night, respectively (see Gibon et al. 2004b). When the
photoperiod was decreased from 12 to 8, 4 and 3 h, the
average rate of starch synthesis increased from 2.6 to 3.3, 4.9
and 4.9 mmol hexose equivalents per gFW per hour, and the
average rate of starch degradation decreased from 2.6 to
1.7, 1.0 and 0.7 mmol hexose equivalents per gFW per hour,
respectively (Fig. 1b).
Despite the lower level of starch at the end of the day and
the decreased rate of starch breakdown during the night,
there was no evidence for a major shortfall of carbohydrates at the end of the night in very short day regimes
(Fig. 2, see also Table 1). The levels of starch and reducing
sugars at the end of the night were similar to those in a
12 h light/12 h dark regime. Sucrose showed a significant
(Table 1) approximately twofold decrease at the end of the
night (Fig. 2). The levels of starch, sucrose and reducing
sugars at the end of the night in short photoperiods were
much higher than after several days of darkness, or at the
end of the night in pgm (Fig. 1).
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
862 Y. Gibon et al.
(a)
45
Extended
night
pgm
12/12
40
Different photoperiod regimes
2/22
3/21
4/20
8/16 12/12
35
30
25
20
15
10
5
0
148 h 72 h 48 h
EL EN
EL EN EL EN
EL EN
EL EN
EL EN
Protein content (mg·gFW−1)
Amino acids (µmol·gFW−1)
Glucose (µmol·gFW−1)
Sucrose (µmol·gFW−1)
Starch in hexose eq. (µmol·gFW−1)
(c)
ED
EN
5
ED
EN
16
Fumarate (µmol·gFW−1)
Malate (µmol·gFW−1)
(b)
4
12
3
2
1
0
8
4
0
2/22
3/21
4/20
8/16
12/12
Photoperiod regime
2/22
3/21
4/20
8/16
12/12
Photoperiod regime
Figure 2. Metabolite levels. Metabolites were measured in wild-type Col-0 rosettes at the end of the light period (EL, white sectors) and
the end of the dark period (ED, grey sectors) in material harvested from plants 17 d after transfer to a 12/12, 8/16, 4/20, 3/21 or 2/22 h
light/h dark photoregime. For comparison, metabolite levels are also shown for wild-type Col-0 that had been grown in a 12 h light/12 h
dark cycle before transfer to continuous darkness for 2, 3 or 7 d, and in the starchless pgm mutant grown in a 12 h light/12 h dark regime
and harvested at the end of the day. (a) Starch, sucrose, reducing sugars, amino acids and protein content. Malate (b) and fumarate
(c) levels are shown separately. The results are the mean ⫾ SE of five replicate samples. The original data are provided in Supporting
Information Table S1. FW, fresh weight.
Amino acid levels increased slightly during the light
period in a 12 h photoperiod (see also Gibon et al. 2006).
Amino acid levels were not affected by shortening the light
period to 8, 4 or 3 h (Fig. 2, Table 1). This contrasts with pgm
where there is a small increase of amino acids, and extended
darkness where there is a threefold increase of amino acids
(Fig. 2, see also Gibon et al. 2006).
Malate and fumarate can accumulate to high levels in
leaves (Chia et al. 2000; Fahnenstich et al. 2007). When Arabidopsis was grown in a 12 h photoperiod, their diurnal
turnover was equivalent to approximately 20–30% of that of
starch (Fig. 2b,c). Short photoperiods led to a two- to threefold decrease of organic acids at the end of the day,decreased
diurnal turnover (from 11.9 to 7.8, 5.7 and 4.2 mmol gFW-1),
an unaltered or slightly increased rate of accumulation in the
light (0.99, 0.98, 1.42 and 1.4 mmol gFW-1 h-1), a much slower
rate of mobilization during the night (from 0.99 to 0.48, 0.28
and 0.20 mmol gFW-1 h-1), and a threefold decrease of the
level at the end of the night (Fig. 2, Supporting Information
Table S1).
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
Adaptation to short photoperiods 863
Fresh weight (FW) basis
Protein basis
Parameter
R
P value
R
P value
Starch (end of night)
Sucrose (end of night)
Glucose (end of night)
Fructose (end of night)
Amino acids (end of night)
Malate (end of night)
Fumarate (end of night)
Malate (end of day)
Fumarate (end of day)
-0.07
-0.76
-0.40
0.78
0.00017
0.090
0.04
-0.73
-0.25
0.87
0.0004
0.29
0.01
-0.86
-0.84
-0.79
-0.93
0.97
0.000002
0.000006
0.00005
0.00000008
0.34
-0.83
-0.86
-0.75
-0.88
0.17
0.00001
0.000002
0.00024
0.0000007
Table 1. Correlation analysis of the
response of carbohydrates, total amino acids
and organic acids to a shortening of the
photoperiod from 12 to 8, 4 and 3 h
Metabolite levels were expressed on an FW or a protein basis. The analysis was carried out
with the values at the end of the night, except for organic acids, where the values at the end
of the day are also analysed. Each analysis is based on five samples for each of the four
photoperiod treatments (n = 20 in total). Parameters that show significant changes
(P < 0.005) are indicated by bold face.
Metabolite levels showed a qualitatively different
response in the extreme 2 h light/22 h dark photoperiod
regime, in which (see earlier discussion) growth was not
possible. Starch synthesis was severely inhibited, leading to
strong reduction of the starch content at the end of the light
period. Organic acid levels at the end of the day decreased
another twofold, compared with the 3 h photoperiod. Unexpectedly, the levels of starch, sugars, amino acids and
organic acids at the end of the night were similar to or
higher than those in a 3 h photoperiod.
Protein content
Total protein decreased by 10–15% as the photoperiod was
decreased from 12 to 3 h (Fig. 2). This decrease was highly
significant (P = 0.00012, Table 2). It was not caused by N
deficiency; amino acids remained unaltered in short photoperiods (see earlier discussion), and nitrate remained unaltered on a FW basis (R = -0.13, P = 0.47) and increased
significantly on a protein basis (R = 0.62, P = 0.0016). There
was also a significant decrease of chlorophyll (Table 2, see
Supporting Information Table S1 for the original data). In
the 2 h photoperiod, protein levels decreased by a further
25% compared with the 3 h photoperiod, and by 35–40%
compared with the 12 h photoperiod.
Protein decreases by about 30% when Col-0 is darkened
for several days (Fig. 2; see also Thimm et al. 2004; Usadel
et al. 2008). In pgm, protein levels are slightly lower than in
wild-type Col-0 (Fig. 2). A more extensive analysis of many
separate experiments confirmed that pgm contains lower
levels of protein than wild-type plants (Hannemann et al.
2009).
Relation between the RGR, starch turnover,
protein content and organic acid levels
Diurnal starch turnover showed a curvilinear relation to the
RGR (Fig. 3a). The rate of starch synthesis was unrelated to
the RGR (data not shown). The rate of starch degradation
was tightly correlated with RGR (Pearson’s R2 = 0.99;
Fig. 3b) and, though less strongly, the protein content
(R2 = 0.86; Fig. 3c). Protein content also correlated with
RGR (R2 = 0.80; Fig. 3d). The average rate of organic acid
mobilization correlated strongly with the rate of starch degradation (R2 = 0.96) and the RGR (R2 = 0.98. not shown),
and more weakly with the protein content (R2 = 0.73, not
shown).
Changes of enzyme activities
Enzyme activities were assayed at the end of the light
period and the end of the night in a 12, 8, 4, 3 and 2 h
photoperiod (see Supporting Information Fig. S1 and Supporting Information Table S1 for the original data). Regression analysis was performed on the combined 12, 8, 4 and
3 h photoperiod data set to identify enzymes that show a
significant change of activity (Table 2). The extreme 2 h
photoperiod treatment was omitted from the regression
analysis. Of the 25 enzymes investigated, 18 showed a significant (P < 0.005) decrease in activity on a FW basis as the
photoperiod was decreased to 3 h. Most others showed a
small but non-significant decrease. Glutamate dehydrogenase (GDH) showed a highly significant increase in activity.
Most enzymes had similar activities at the end of the
night and the end of the day. Marked diurnal changes in
activity were found for ADP-glucose pyrophosphorylase
(AGPase) and nitrate reductase (NR). Regression analyses
were therefore performed separately on the data sets for
the end of the night and the end of the day. Short photoperiods led to a significant decrease of AGPase activity at the
end of the day but not at the end of the night. Short photoperiods led to a significant decrease of NR activity at the
end of the night but not at the end of the day.
Comparison of the changes of enzyme
activities and the overall protein content
In many cases, the decrease of the enzyme activity
resembled the decrease of the total protein content
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
864 Y. Gibon et al.
Fresh weight
(FW) basis
Protein basis
Parameter
R
P value
R
P value
Protein
Chlorophyll
Ribulose 1·5-bisphosphate carboxylase/
oxygenase
Phosphoglycerokinase
NADP-dependent glyceraldehyde-3-phosphate
dehydrogenase
Aldolase
Transketolase
Plastidic phosphoglucoisomerase
AGPase (end of night)
AGPase (end of day)
Cytosolic phosphoglucoisomerase
Phosphoglucomutase
Sucrose phosphate synthase
Acid invertase
Pyrophosphate-dependent phosphofructokinase
ATP-phosphofructokinase
Pyruvate kinase
Phosphoenolpyruvate carboxylase
Citrate synthase
Isocitrate dehydrogenase
Fumarase
Malate dehydrogenase
Nitrate reductase (NR) (end of night)
NR (end of day)
Glutamine synthetase
Ferredoxin-dependent glutamate synthase
Glutamate dehydrogenase
-0.57
-0.80
-0.50
0.00012
0.000000001
0.0011
–
-0.58
-0.28
–
0.00015
0.087
-0.39
-0.31
0.014
0.054
-0.10
-0.10
0.54
0.53
-0.55
-0.47
-0.14
-0.33
-0.83
-0.56
-0.50
-0.46
-0.29
-0.66
-0.69
-0.58
-0.66
-0.51
-0.56
-0.7
-0.53
-0.75
0.20
-0.08
-0.35
0.58
0.00026
0.0038
0.40
0.20
0.000081
0.00022
0.0013
0.0029
0.064
0.000006
0.000001
0.00009
0.0000005
0.00095
0.00018
0.0000008
0.00051
0.00034
0.40
0.65
0.027
0.00027
-0.25
-0.11
0.11
0.13
0.75
-0.21
-0.06
-0.09
0.03
-0.41
-0.47
-0.31
-0.52
-0.08
-0.38
-0.51
-0.15
-0.14
0.46
0.20
-0.07
0.69
0.12
0.53
0.53
0.58
0.00083
0.19
0.73
0.53
0.88
0.011
0.0024
0.0050
0.0007
0.63
0.016
0.00088
0.37
0.56
0.0048
0.22
0.67
0.000005
Table 2. Correlation analysis of the
response of protein, chlorophyll and enzyme
activities to a shortening of the photoperiod
from 12 to 8, 4 and 3 h
Protein was expressed on an FW basis, and chlorophyll and enzyme activities on an FW or
a protein basis before the analysis. For all parameters except ADP-glucose pyrophosphorylase (AGPase) and NR, the values at the end of the night and the end of the day were pooled
(n = 10 for each of the four photoperiod treatments, 40 in total). For AGPase and NR, the
data for the end of the night and the end of the day were analysed separately (n = 20 in total).
Parameters that show significant changes (P < 0.005) are indicated by bold face.
(Table 2; compare also Fig. 2a and Supporting Information
Fig. S1). To identify enzymes that show changes in activity
that are not just because of the changes in the total protein
content, we recalculated the activities on a protein basis
(Fig. 4), and performed regression analysis on the combined
12, 8, 4 and 3 h photoperiod data set (Table 2).
There was no significant change in the activity of ribulose
1·5-bisphosphate carboxylase/oxygenase (Rubisco; Fig. 4a)
and four other Calvin cycle enzymes (Fig. 4b–e) when the
photoperiod was shortened from 12 to 8, 4 and 3 h. There
was a decrease of Rubisco, aldolase and transketolase activity on a protein basis in the extreme 2 h photoperiod. In the
pathway of starch synthesis, pPGI activity did not show any
significant changes (Fig. 4f). AGPase remained high at the
end of the night, but showed a strong and significant
decrease at the end of the light period (Fig. 4g). The diurnal
changes of AGPase activity became increasingly marked as
the photoperiod was shortened. Cytosolic PGI (cPGI),
phosphoglucomutase (PGM) and sucrose phosphate synthase are required for sucrose synthesis. Their activities
remained high in very short day treatments (Fig. 4h–k).
Figure 4l–o shows enzymes from glycolysis including
ATP-phosphofructokinase (PFK), pyrophosphate (PPi)dependent phosphofructokinase (PFP), pyruvate kinase
(PK) and phosphoenolpyruvate carboxylase (PEPCase),
and Fig. 4p–s shows citrate synthase (CS), NAD-isocitrate
dehydrogenase (ICDH), fumarase and NAD-malate dehydrogenase (MDH), which are involved in organic acid
metabolism. There was a marked and significant decrease
of PEPCase activity (Fig. 4o, P = 0.0007, see Table 2), and
a weaker but significant (see Table 2) decrease of PFK
(Fig. 4m), PK (Fig. 4n), ICDH (Fig. 4q) and fumarase
(Fig. 4r) activity in short photoperiods, whereas PFP
(Fig. 4l), CS (Fig. 4p) and NAD-MDH (Fig. 4s) activities
did not show any significant change. In nitrogen metabolism, NR activity did not show any significant changes at the
end of the night but increased significantly at the end of
the day in short photoperiods (Fig. 4t). As for AGPase, the
diurnal changes become more marked in short photoperiods. In the extreme 2 h photoperiod, NR activity remained
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
Adaptation to short photoperiods 865
Rate of starch degradation
(mmol hexose·gFW−1·h−1)
(c)
(a)
0.10
0.05
10
20
30
0.15
RGR (gFW·gFW−1·d−1)
0
Diurnal starch turnover
(mmol hexose·gFW−1 each day)
RGR (gFW·gFW−1·d−1)
R 2 = 0.861
1
0
0
5
10
15
20
Protein content (mg·gFW−1)
0.15
0
(b)
2
(d)
0.10
R 2 = 0.799
0.05
0
0.10
0
5
10
15
20
Protein content (mg·gFW−1)
(e)
R 2 = 0.993
0.05
0
0
1
2
3
Rate of starch degradation
(mmol hexose·gFW−1·h−1)
Organic acid mobilization
(mmol ·gFW−1·h−1)
RGR (gFW·gFW−1·d−1)
0.15
3
1.0
R 2 = 0.966
0.5
0
0
1
2
3
Rate of starch degradation
(mmol hexose·gFW−1·h−1)
Figure 3. Relative growth rate (RGR), starch metabolism and the protein content. (a) Diurnal starch turnover and RGR. (b) Starch
breakdown rate and RGR. (c) Starch breakdown rate and the overall protein content. (d) RGR and the overall protein content.
(e) Starch breakdown rate and organic acid mobilization at night. The results are calculated from the data in Figs 1 and 2. FW, fresh weight.
low at the end of the day. Glutamine synthase (GS) and
ferredoxin-dependent glutamate synthase (Fd-GOGAT)
activity remained unaltered or increased in short days,
except for the 2 h treatment (Fig. 4u,v). GDH activity
increased progressively and significantly in short photoperiods (Fig. 4w).
Overall, these measurements show that when activity
is expressed on a protein basis, the activities of enzymes
that are required for photosynthesis, sucrose and starch
synthesis and inorganic nitrogen assimilation remain high
whereas several enzymes from glycolysis and organic acid
metabolism still show a significant decrease of their activity
in short photoperiods.
Comparison of enzyme activities in short
photoperiods with those found in an extended
night or the starchless pgm mutant
The data set for enzyme activities in different photoperiods
was combined with the published data sets for the
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
866 Y. Gibon et al.
500
(a)
6000
400
300
2000
100
2
3
4
8
12
Phosphoglucose
isomerase (plastid)
(f)
30
0
120
100
2
3
4
8
12
2
3
4
8
12
600
60
400
500
100
0
0
2
3
4
8
12
Phosphofructokinase (PPi)
(l)
400
400
200
300
200
100
0
0
2
3
4
8
12
16
2
3
4
8
500
200
100
2
3
4
8
12
12
Phosphoglucomutase
(i)
Sucrose phosphate
synthase
160
(j)
120
3
4
8
12
Citrate synthase
(p)
2
3
4
8
12
Phosphofructo
kinase (ATP)
(m)
8
0
100
20
0
0
2
3
4
8
12
Isocitrate DH (NAD)
(q)
14
12
2
3
4
8
12
Pyruvate kinase
(n)
3
4
8
12
Nitrate reductase
(t)
300
6
200
2
3
4
8
12
Glutamine
synthetase
(u)
0
120
100
60
40
2
3
4
8
12
Fumarase
0
6000
(r)
5000
2
3
4
8
12
PEP carboxylase
(o)
2
3
4
8
12
Malate DH (NAD)
(s)
4000
2000
3
4
8
12
2
3
4
8
12
Fd-GOGAT
(v)
0
60
50
20
10
0
2
1000
30
0
12
Photoperiod length
Acid invertase
(k)
10
40
0
8
50
40
20
4
60
60
20
3
0
80
10
2
50
End of night
20
100
30
20
60
End of light
3000
80
40
500
8
100
12
40
400
2
2
8
30
4
0
4
10
40
4
4
3
20
60
10
12
2
30
40
8
2
0
40
80
2
1
Transketolase
AGPase
12
3
50
300
80
4
0
600
200
20
16
400
(g)
300
40
0
800
20
0
700
(e)
600
40
Phosphoglucose
isomerase (cytosol)
80 (h)
5
FBP-aldolase
(d)
60
10
6
500
80
20
0
GAPDH (NADP)
(c)
4000
200
0
1000
nmol·mg−1 protein·min−1
600
Phosphoglycerokinase
8000
(b)
Rubisco
2
3
4
8
12
0
2
3
4
8
12
Glutamate DH
(w)
2
3
4
8
12
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
Adaptation to short photoperiods 867
Figure 4. Enzyme activities on a protein basis. Enzyme activities were measured at the end of the light period (open bars) and the end
of the night period (solid bars) in wild-type Arabidopsis growing in a 12/12, 8/16, 4/20, 3/21 or 2/22 h light/dark cycle. The data on a fresh
weight basis are shown in Supporting Information Fig. S1. The protein levels from Fig. 2 were used to recalculate all activities on a leaf
protein basis. (a) Ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco). (b) Phosphoglycerokinase. (c) NADP-dependent
glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH). (d) Fructose-bisphosphate (FBP) aldolase. (e) Transketolase. (f) Plastid
phosphoglucose isomerase. (g) ADP-glucose pyrophosphorylase (AGPase). (h) Cytosolic phosphoglucose isomerase.
(i) Phosphoglucomutase. (j) Sucrose phosphate synthase. (k) Acid invertase. (l) Pyrophosphate (PPi)-dependent phosphofructokinase.
(m) ATP-dependent phosphofructokinase. (n) Pyruvate kinase. (o) Phosphoenolpyruvate (PEP) carboxylase. (p) Citrate synthase.
(q) NAD-dependent isocitrate dehydrogenase (DH). (r) Fumarase. (s) NAD-dependent malate DH. (t) Nitrate reductase. (u) Glutamine
synthetase. (v) Ferredoxin-dependent glutamate synthase (Fd-GOGAT). (w) Glutamate dehydrogenase (Glutamate DH). The results are
the mean ⫾ SE (n = 5 separate samples).
responses in prolonged darkness and in the starchless pgm
mutant (Gibon et al. 2004b), and subjected to correlation
and clustering analysis. Whereas prolonged darkness leads
to a sustained C depletion, pgm alternates every day
between high and very low sugar (see Introduction). The
combined data set included a total of 17 treatments; the end
of the light period and the end of the night for the five
photoperiod treatments with Col-0, the end of the light
period and the end of the night for pgm, and 48, 72 and
148 h of prolonged darkness with Col-0. It contained 16
enzymes, which had been measured in all of these treatments. The enzyme activities were expressed on a FW basis,
because normalization on protein leads to loss of the activity changes of many of the enzymes. Activities were normalized on the average of the activity at the end of the light
period and the end of the night in wild-type Col-0 in a 12 h
photoperiod treatment from the same experiment.
Table 3 summarizes Pearson’s regression coefficients (R)
between the different treatments. As expected, the profiles
in wild-type Col-0 in a 12 h photoperiod differ from the
Table 3. Regression coefficients between the enzyme activities
in very short days, and enzyme activities after 6 d extended
darkness, or in starchless pgm mutants
pgm EL
pgm EL
pgm EN
148 h XN
48 h XN
12 h L/12 h N EL
12 h L/12 h N EN
8 h L/8 h N EL
8 h L/8 h N EN
4 h L/4 h N EL
4 h L/4 h N EN
3 h L/3 h N EL
3 h L/3 h N EN
2 h L/2 h N EL
2 h L/2 h N EN
pgm EN
148 h XN
48 h XN
0.77**
0.24
0.74**
0.14
0.62**
0.89**
–
0.77**
0.24
0.14
0.07
-0.08
0.64**
0.41
0.41
0.53*
0.63**
0.00
0.78**
0.41
–
0.74**
0.62**
0.13
-0.07
0.47
0.38
0.38
0.48
0.40
0.14
0.59*
0.56*
–
0.89**
0.29
-0.20
0.05
0.31
0.16
0.24
0.01
0.31
0.27
0.56**
–
0.39
-0.29
-0.03
0.24
0.00
0.09
-0.08
0.32
0.23
0.38
Pearson regression coefficients (R) and P values (**, less than 0.01;
*, less than 0.05; both indicated in bold face) were calculated with
the responses of 16 enzymes to the various treatments. All enzyme
activities were log2 normalized on the level in wild-type Col-0 at the
end of the night in a 12 h light/12 h dark cycle.
D, dark; EL, end of the light period; EN, end of the night period; L,
light; XN, extended night.
profiles in prolonged darkness and in pgm. When the
photoperiod is shortened to 8, 4 or 3 h, the wild-type Col-0
profile at the end of the light period becomes increasingly
similar to that in pgm at the end of the light period
(R = 0.41–0.78, P < 0.01).When the profiles are compared at
the end of the night the similarity is weaker and nonsignificant (R = 0.14–0 56). The similarity to the profile in
prolonged darkness is also weaker and non-significant
(R < 0.16 and <0.36 for profiles from the photoperiod treatments at the end of the light period and the end of the dark
period, respectively). When the photoperiod is shortened to
2 h, the similarity to pgm increases further (R = 0.41–0.78,
P < 0.01 at the end of the light period and P < 0.05 at the
end of the night). A significant correlation also appears
between the profiles at the end of the night and after 148 h
prolonged darkness (R = 0.56, P < 0.01).
Figure 5 shows an unbiased two-way clustering of these
17 experimental treatments against the profile for the 16
enzyme activities and the total protein content. The protein
content and enzyme activities are shown as a heat map. The
photoperiod treatments separate along the axis from right
to left, according to the length of the photoperiod. This
presumably reflects the magnitude of the response of the
enzyme activities, which becomes progressively larger as
the photoperiod is shortened. The end of the night and the
end of the day treatments group together for a given photoperiod. This shows that the long-term adjustments of
enzyme activities to the photoperiod are larger than the
diurnal changes in a given photoperiod.The only exceptions
are the 3 and 4 h photoperiods, where the samples group
according to the time of harvest, probably because of the
more accentuated changes of AGPase and NR activities
across the day–night cycle. The profiles of wild-type plants
grown under photoperiods ranging from 8 to 3 h are closer
to the pgm profiles than to the profiles of plants after an
extension of the night. The largest linkage distance was
found for the 2 h photoperiod treatment.
Figure 5 also provides an overview of the responses of
individual parameters. Total protein decreases in all three
treatments. GDH activity rises in all of the treatments; this
is summarized in Fig. 6, which emphasizes that the increase
in activity in the short photoperiod treatments is much
smaller than in pgm or prolonged darkness. Most other
enzyme activities decrease in all three treatments (see also
Supporting Information Fig. S3). Rubisco, SPS, PEPC, PK
and fumarase activity show a larger increase in very short
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
868 Y. Gibon et al.
WT-E
EL-2 h
WT-E
EN-2 h
WT-72 h XN
WT-148 h XN
WT-48 h XN
N-12 h
pgm-EN
pgm-EL
L-12 h
WT-E
EL-3 h
WT-E
EL-4 h
WT-E
EN-3 h
WT-E
EN-4 h
WT-E
EL-8 h
WT-E
EN-8 h
WT-EL
L-12 h
WT-EL
L-12 h
N-12 h
WT-EN
WT-EN
N-12 h
Glutamine synthetase
Nitrate reductase
Acid invertase
Glutamate DH
AGPase
Fructose-1,6-bisphosphatase (cytosolic)
Fd-GOGAT
GAPDH (NADP)
Phosphofructokinase (PPi)
ShikimateDH
Transketolase
Fumarase
PEP carboxylase
Rubisco
Sucrose phosphate synthase
Pyruvate kinase
Protein content
−3
0
3
Log2 ratio to control
GDH activity relative to average
in a 12/12 h cycle (log2 scale)
Figure 5. Unbiased two-way clustering of treatments against 16 enzyme activities and protein. The treatments are wild-type (WT) Col-0
grown in a 12/12, 8/16, 4/20, 3/21 and 2/22 h light/h dark cycle, pgm grown in a 12 h light/12 h dark light / dark cycle and wild-type Col-0
harvested 48, 72 and 148 h into an extended night. The dotted lines demarcate the pgm and extended night treatments. All enzyme
activities were normalized on the level in wild-type Col-0 at the end of the night in a 12 h light/12 h dark cycle. The normalized values are
shown on a false colour scale (see figure). AGPase, ADP-glucose pyrophosphorylase; L, light; D, dark; DH, dehydrogenase; EL, end of the
light period; EN, end of the night period; Fd-GOGAT, ferredoxin-dependent glutamate synthase; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; PEP, phosphoenolpyruvate; XN, extended night.
Extended
night
1.4
pgm
12/12
1.2
Different photoperiod regimes
2/22
3/21
4/20
EL EN EL EN
EL EN
8/16 12/12
1.0
0.8
0.6
0.4
0.2
0.0
−0.2
−0.4
148 h 72 h 48 h
EL EN
EL EN
EL EN
Figure 6. Comparison of glutamate dehydrogenase (GDH) activity in short photoperiods, in prolonged darkness and in the starchless
pgm mutant. GDH activity was measured in wild-type Col-0 rosettes at the end of the light period (EL, white sectors) and the end of the
dark period (EN, grey sectors) in material harvested from plants 17 d after transfer to a 12/12, 8/16, 4/20, 3/21 or 2/22 h light/h dark
photoregime. For comparison, GDH activity is shown for wild-type Col-0 grown in a 12 h light/12 h dark cycle before transfer to
continuous darkness for 2, 3 or 7 d, and in the starchless pgm mutant grown in a 12 h light/12 h dark regime and harvested at the end of
the day (EL, white sectors) and night (EN, grey sectors). The results are the mean ⫾ SE of five replicate samples.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
Adaptation to short photoperiods 869
photoperiods and in prolonged darkness than in pgm. In
specific cases, there is a qualitative difference between the
treatments. Invertase activity does not increase in short
photoperiods, even though it rises in pgm and prolonged
darkness. AGPase activity decreases in the short photoperiod and pgm treatments, but rises slightly in prolonged
darkness.As already noted,AGPase activity decreases in the
light and peaks at the end of the night (see also Gibon et al.
2004a,b). NR shows only small changes in short photoperiods and pgm, but a large decrease in prolonged darkness. NR
activity typically increases in the first 2 h of the light period in
a 12 h photoperiod (Gibon et al. 2004b). The high AGPase
and low NR activity in prolonged darkness may be caused by
the absence of a light phase in this treatment.
DISCUSSION
The experiments presented in this article investigate: (1)
whether Arabidopsis is able to adjust to a large decrease in
the C supply and grow in extremely short photoperiods;
(2) if changes in starch turnover, the protein content and
central metabolism contribute to the adjustment to low
C; and (3) whether the response to short photoperiods
resembles the responses when C deficiency is imposed by
two other treatments; a severe sustained C starvation after
darkening wild-type Col-0 for several days, and a recurring
transient C limitation in the starchless pgm mutant.
Adjustment of growth, starch turnover
and the protein concentration to very
short photoperiods
Wild-type Arabidopsis grows in extremely short photoperiods, down to a 3 h light/21 h dark cycle, showing that
it adjusts flexibly to very large changes in the C supply.
One contributory factor is a major modification of starch
turnover. In short photoperiods, starch is synthesized more
rapidly in the light and is degraded more slowly during the
night. This resembles the response previously documented
in less extreme photoperiods (see Introduction).As a result,
the amount of starch and the levels of sugars and amino
acids at the end of the dark period are largely independent
of the length of the light period.
Short photoperiods also lead to a significant 10–15%
decrease of the rosette protein concentration. This decrease
is unlikely to be caused by a direct lack of amino acids. The
activities of enzymes involved in the assimilation of inorganic nitrogen remained high or increased (see below for
more discussion), and nitrate and amino acid levels
remained high in short photoperiods. A similar decrease of
the protein concentration in short photoperiods has been
seen in many other independent experiments, where the
photoperiod was varied between 2 and 20 h (Hannemann
et al. 2009).
Expression profiling in wild-type Col-0 has revealed that
diurnal changes of C lead to coordinated changes in the
levels of transcripts for hundreds of genes that are involved
in protein synthesis and targeting, with transcripts starting
to decrease early in the night (Bläsing et al. 2005; Usadel
et al. 2008). Further, polysome loading and, by implication,
the rate of protein synthesis decreases during the night
(Kumar, Piques & Stitt, unpublished data). The simplest
explanation for the decreased protein content in short day
conditions is that C regulates the rate of protein synthesis.
However, it is possible that light-signalling also plays a role.
Very short days (2–4 h) lead to petiole elongation (data not
shown), which is reminiscent of the responses to low light.
Kozuka et al. (2005) showed that petiole extension and leaf
blade expansion is regulated by an interaction between
sugars and phytochrome and cryptochrome-dependent
light signalling, whereas Nozue & Maloof (2006) have proposed that hypocotyl growth is regulated by an interaction
between C signalling, light signalling and diurnal responses.
Our analyses reveal a striking positive correlation
between the rate of starch breakdown and the RGR
(R2 = 0.97, Fig. 3e).This relation has been confirmed in independent experiments (Hannemann et al. 2009). This result
implies that starch turnover and growth are tightly coordinated. More studies are needed to explore the causal reasons
for this tight correlation. One contributing factor may be the
decrease of the protein content in short photoperiods.There
was a correlation between the protein content and the rate of
starch breakdown (R2 = 0.86, Fig. 3c). This correlation has
been confirmed in a meta-analysis of data from 27 experiments (R = 0.55, P = 4.4 ¥ 10-15, Hannemann et al. 2009). A
lower protein content will decrease the requirement for C in
two ways. Firstly, a decrease in the protein content, or more
generally the nitrogen content, is typically accompanied by a
near-linear decrease in the rate of respiration (James 1953;
Ryan 1991; van der Werf et al. 1992). This will decrease the
amount of starch that is required to support respiration
during the night. Secondly, a decrease in the protein content
may decrease construction costs, that is, the amount of C that
is needed to produce a unit amount of biomass. The assimilation of inorganic nitrogen into amino acids and the subsequent conversion of amino acids to protein are energetically
expensive processes, with both requiring about five ATPs per
amino acid incorporated into protein. Modelling and experiments that use cycloheximide to inhibit protein synthesis
indicate that protein synthesis is responsible for 20–70% of
the total respiratory costs in plant tissues (Penning de Vries
1975; van der Werf et al. 1992; Bouma et al. 1994; Zagdanska
1995; Noguchi, Nakajima & Terashima 2001; Hachiya,
Treashima & Nochuchi 2007). The observation that plant
growth often occurs during the night (Schurr, Walter &
Rascher 2006; Wiese et al. 2007) underlines the potential
importance of a link between the C supply from starch
degradation and construction costs. This is likely to become
increasingly important in very short photoperiods.
Rasse & Tocquin (2006) recently modelled the relation
between photosynthesis, starch turnover, sugar levels, respiration and growth. Their model assumes that starch is
produced at a baseline rate when photosynthate is low, and
does not address the question of how plants adjust to
changes in the C supply. Our and (see Introduction) earlier
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
870 Y. Gibon et al.
studies show that starch synthesis is increased when less C is
available. Our study also shows that the changes in starch
turnover are accompanied by coordinated changes in the
protein content and the rate of growth. It will be important in
the future to model these additional interactions, and quantitatively explore their impact on C balance and growth.
Adjustment of central metabolism to very
short photoperiods
Enzyme activities and metabolite levels were measured to
investigate whether changes in central metabolism contribute to the adjustment to short photoperiods. Shortening the
photoperiod from 12 to 3 h led to a relatively small decrease
in the activities of enzymes that are required for C fixation
and starch and sucrose synthesis. The decrease was largely
caused by a decrease of the overall protein content. This
response will minimize the inhibition of photosynthesis and
carbohydrate synthesis, and maintain C gain in short photoperiods. The activities of enzymes involved in nitrogen
assimilation including NR, GS and Fd-GOGAT also
remained high. Indeed, NR activity at the end of the light
period actually increased in short photoperiods. Amino acid
levels also remained high in short photoperiods. This highlights the ability of Arabidopsis to maintain a metabolic
balance in short days.There is a marked contrast to tobacco,
where growth in an 8 h photoperiod led to a strong inhibition of NR activity, a marked decrease of amino acids and a
large decrease of leaf protein (Matt et al. 1998).
AGPase showed marked diurnal responses, with activity
increasing during the night and decreasing in the light
period. These diurnal changes were absent in a 12 h photoperiod, and became larger as the photoperiod became
shorter. Gibon et al. (2004a,b) also reported that diurnal
changes of AGPase activity are absent in Col-0 in a 20 h,
small in a 12 h photoperiod and marked in a 7 h photoperiod, whereas in pgm diurnal changes are already seen in a
12 h photoperiod, indicating that they are a response to
change of C rather to the light period per se. The diurnal
changes of AGPase activity are caused by the changes of
AGPase protein (Gibon et al. 2004b). High activity of
AGPase at the end of the night might contribute to the high
rate of starch synthesis in the following light period (see
earlier discussion). The mechanisms that lead to these
diurnal changes are unknown.
Eight of the enzymes that we investigated are involved
in glycolysis and respiration. Of these, five (ATPphosphofructokinase, pyruvate kinase, PEPCase, isocitrate
dehydrogenase and fumarase) decreased more strongly
than the total protein content in short photoperiods. The
decrease was especially marked for PEPCase, which is
required for net production of organic acids. In agreement,
organic acids decreased much more strongly than carbohydrates and amino acids in short photoperiods.
Low levels of organic acids might serve to decrease C
utilization for respiration at night. Respiration of malate
requires the concerted action of NADH-MDH and malic
enzyme. The latter generates pyruvate, which is converted
to acetyl-CoA and combines with the oxaloacetate
generated by NADH-MDH. Fahnenstich et al. (2007, 2008)
recently showed that over-expression of NADP-malic
enzyme in Arabidopsis leads to lower levels of malate and
fumarate. This was associated with faster senescence in
continuous darkness, and pale green leaves and increased
accumulation of starch when the plants were grown in low
irradiance and short day conditions, supporting the idea
that organic acids provide an important respiratory substrate when carbohydrates are low. Fahnenstich et al. (2008)
proposed that starch synthesis is stimulated when less C is
stored in organic acids. In our experiments, short photoperiods led to a twofold increase in the rate of starch accumulation in the light but did not markedly affect the rate of
malate and fumarate accumulation. On the other hand, the
rates of starch degradation and organic acid utilization
during the night correlated with each other and with RGR.
This indicates that although allocation to these two C stores
may be differentially regulated, their utilization is tightly
coordinated.
Response to a very short 2 h photoperiod
Arabidopsis Col-0 was unable to grow in a 2 h photoperiod,
but did survive for several weeks. There were several
marked changes in metabolism, compared with a 3 h photoperiod. Firstly, there was a further and marked decrease
in total protein. Secondly, there was a marked decrease of
NR, GS and Fd-GOGAT activity. These three enzymes are
required for inorganic nitrogen assimilation. The reduction
of NR was in part because activity no longer rose during the
light period, and is reminiscent of the inhibition of NR
activity in tobacco in an 8 h photoperiod (see earlier discussion).Thirdly, starch synthesis was inhibited.The reasons for
this unexpected inhibition are unclear, as similar AGPase
activities were found at the end of the night in a 3 and a 2 h
photoperiod. Finally, and most unexpectedly, the levels of
starch, sugars and amino acids at the end of the night in a 2 h
photoperiod were similar to those at the end of the night in
longer photoperiods.These results indicate that the inability
to grow in a 2 h photoperiod is not a simple consequence of
an acute lack of sugars or amino acids. Rather, it appears to
be because of an inhibition of the utilization of these central
metabolites for growth. Further studies will be needed to
reveal the precise reason for this inhibition.
Comparison of the response of enzyme activities
to short photoperiods or prolonged darkness in
wild-type Col-0 and to repeated transient
periods of C starvation in pgm
There were several similarities between the enzyme activity
profiles in short photoperiods and the profiles in prolonged
darkness or the starchless pgm mutant. All the three treatments led to a decrease of the overall protein content, lower
activities on a FW basis of many enzymes from photosynthetic C metabolism, nitrogen assimilation, glycolysis and
respiration, and an increase of GDH activity. Regression
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
Adaptation to short photoperiods 871
analysis and two-way clustering indicated that the
enzyme profile in 8, 4 or 3 h photoperiods resembles the
profile in pgm. This is consistent with the proposal (see
Introduction) that diurnal changes of transcripts are integrated over time as changes of the protein content and
enzyme activities.
All the three C-depletion treatments led to an increase of
GDH activity, indicating that high GDH activity is a good
metabolic marker for C deprivation. GDH operates in
the direction of ammonium release (Miflin & Habash 2002),
and recycles glutamate to 2-oxoglutarate during amino
acid catabolism (Melo-Oliveira, Oliveira & Coruzzi 1996;
Lam et al. 2001). As already noted, carbohydrate levels at
the end of the night in short photoperiods were higher than
at the end of the night in pgm, or in prolonged darkness.
This indicates that adjustments of starch turnover and
metabolism allow Arabidopsis to avoid severe C depletion
when it is grown in short photoperiods. In agreement, GDH
activity did not increase as much in short photoperiods as in
prolonged darkness or in pgm. The increase of GDH activity in low-C conditions may be partly caused by transcriptional regulation. GDH is encoded by a small family of
three genes. Two of these (GDH1, GDH2) are strongly
induced by C starvation in seedlings and rosettes (Price
et al. 2004; Thimm et al. 2004; Osuna et al. 2007; Usadel et al.
2008). In rosettes, transfer to prolonged darkness leads to a
rapid increase of GDH1 and GDH2 transcripts, whereas
GDH activity increases gradually for several days (Gibon
et al. 2004b). In pgm, GDH1 and GDH2 transcripts peak
transiently in the night, whereas GDH activity is stable and
about twice that in wild-type Col-0.
In conclusion, Arabidopsis Col-0 adjusts to increasingly
short photoperiods by a progressive inhibition of growth,
stimulation of starch synthesis, inhibition of starch degradation and decrease of the overall protein content. There is
a striking correlation between the rate of starch breakdown and the rate of growth, and a significant correlation
between both of these parameters and the overall protein
concentration.The activities of enzymes that are involved in
respiration decrease, relative to enzymes that are involved
in photosynthesis, starch and sucrose synthesis and nitrogen
assimilation. This allows C levels to be maintained at the
end of the night that are similar to those in longer photoperiods, and are much higher than in acute or regularly
recurring C starvation. These results highlight the ability of
Arabidopsis to restrict C utilization and gauge growth to
the C supply, and point to the importance of regulatory
mechanisms that regulate starch turnover and the protein
concentration as important components of this response.
ACKNOWLEDGMENTS
This research was supported by the Max Planck Society and
by the German Ministry for Research and Technology, in
the framework of the German Plant Genomics programme
GABI (0312277A, 0313110, 0313123) and GoFORSYS
(http://www.goforsys.de/). We are grateful to Kristin Retzlaff for validating the PGI assay. The writing of the paper
was greatly helped by perceptive comments from two
anonymous referees.
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Received 25 November 2008; received in revised form 28 January
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Enzyme activities calculated on a fresh weight
basis.Enzyme activities were measured at the end of the light
period (open bars) and end of the night period (solid bars)
in wild-type Arabidopsis growing in a 12/12, 8/16, 4/20, 3/21
or 2/22 h light/dark cycle. (a) Ribulose 1·5-bisphosphate
carboxylase/oxygenase (Rubisco). (b) Phosphoglycerate
kinase. (c) NADP-dependent glyceraldehyde-3-phosphate
dehydrogenase
(NADP-GAPDH).
(d)
Fructosebisphosphate (FBP)-aldolase. (e) Transketolase. (f) Plastid
phosphoglucose isomerase (g) Phosphoglucomutase. (h)
ADP-glucose pyrophosphorylase (AGPase). (i) Cytosolic
phosphoglucose isomerase. (j) Sucrose phosphate synthase.
(k) Acid invertase. (l) Pyrophosphate-dependent phosphofructokinase. (m) ATP-dependent phosphofructokinase. (n)
Pyruvate kinase. (o) Phosphoenolpyruvate (PEP) carboxylase. (p) Citrate synthase. (q) NAD-dependent isocitrate
dehydrogenase. (r) Fumarase. (s) NAD-dependent malate
dehydrogenase. (t) Nitrate reductase. (u) Glutamine
synthetase. (v) Ferredoxin-dependent glutamate synthase
(GOGAT). (w) Glutamate dehydrogenase (glutamate DH).
The results are the mean ⫾ SE. (n = 5 separate samples).
Figure S2. Comparison of the changes of enzyme activity
in pgm and in wild-type Col-0 after 7 d of extended darkness. The plot is recalculated from data in Gibon et al.
(2004b).
Figure S3. Comparison of the activity of enzymes from the
Calvin cycle, glycolysis, the tricarboxylic acid cycle, sucrose
and starch synthesis and nitrogen metabolism in short
photoperiods, in prolonged darkness and in the starchless
pgm mutant. Rubisco, NADP-dependent glyceraldehyde-3phosphate dehydrogenase (NADP-GAPDH), transketolase, AGPase, sucrose phosphate synthase, pyrophosphate
(PPi)-dependent phosphofructokinase, ATP-dependent
phosphofructokinase, pyruvate kinase, phosphoenolpyruvate (PEP) carboxylase, fumarase, ferredoxin-dependent
glutamate synthase, glutamine synthetase and nitrate reductase activities were measured in wild-type Col-0 rosettes at
the end of the light period (EL, white sectors) and the end of
the dark period (EN, grey sectors) in material harvested
from plants 17 d after transfer to a 12/12, 8/16, 4/20, 3/21 or
2/22 h light/h dark photoregime. For comparison, the same
enzyme activities are shown for wild-type Col-0 grown in a
12 h light/12 h dark cycle before transfer to continuous darkness for 2, 3 or 7 d, and in the starchless pgm mutant grown in
a 12 h light/12 h dark regime and harvested at the end of the
day (EL, white sectors) and night (EN, grey sectors). The
results are the mean ⫾ SE of five replicate samples.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874
874 Y. Gibon et al.
Table S1. Metabolite levels and enzyme activities in wildtype Col-0 grown in a 12 h light/12 h dark, a 8 h light/16 h
dark, a 4 h light/20 h dark, a 3 h light/21 h dark and a 2 h
light/22 h dark photoperiod, in the starchless pgm mutant
grown in a 12 h light/12 h dark photoperiod, or in wild-type
Col-0 subjected to a 48, 72 or 148 h extension of the night.
The data are provided as means ⫾ SE of five replicate
samples.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supporting materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 859–874