Plant Physiol. (1998) 118: 149-158
Chilling Delays Circadian Pattern of Sucrose Phosphate Synthase
and Nitrate Reductase Activity in Tomato1
Tamara L. Jones2,
Dawn E. Tucker, and
Donald R. Ort*
Department of Plant Biology (T.L.J., D.E.T., D.R.O.) and
Photosynthesis Research Unit, United States Department of
Agriculture/Agricultural Research Service (D.R.O.), University of
Illinois, Urbana, Illinois 61801-3838
 |
ABSTRACT |
Overnight
low-temperature exposure inhibits photosynthesis in chilling-sensitive
species such as tomato (Lycopersicon esculentum) and
cucumber by as much as 60%. In an earlier study we showed that one
intriguing effect of low temperature on chilling-sensitive plants is to
stall the endogenous rhythm controlling transcription of certain
nuclear-encoded genes, causing the synthesis of the corresponding
transcripts and proteins to be mistimed when the plant is rewarmed.
Here we show that the circadian rhythm controlling the activity of
sucrose phosphate synthase (SPS) and nitrate reductase (NR), key
control points of carbon and nitrogen metabolism in plant cells, is
delayed in tomato by chilling treatments. Using specific protein kinase
and phosphatase inhibitors, we further demonstrate that the
chilling-induced delay in the circadian control of SPS and NR activity
is associated with the activity of critical protein phosphatases. The
sensitivity of the pattern of SPS activity to specific inhibitors of
transcription and translation indicates that there is a
chilling-induced delay in SPS phosphorylation status that is caused by
an effect of low temperature on the expression of a gene coding for a
phosphoprotein phosphatase, perhaps the SPS phosphatase. In contrast,
the chilling-induced delay in NR activity does not appear to arise from
effects on NR phosphorylation status, but rather from direct effects on
NR expression. It is likely that the mistiming in the regulation of SPS
and NR, and perhaps other key metabolic enzymes under circadian
regulation, underlies the chilling sensitivity of photosynthesis in
these plant species.
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INTRODUCTION |
Most warm-climate plant species are sensitive to brief exposures
to low, nonfreezing temperatures. Low-temperature exposure in
combination with high irradiance causes rapid, often very severe inhibition of photosynthesis in a broad range of plants, including maize (Baker et al., 1983
), cucumber (Peeler and Naylor, 1988), and
tomato (Lycopersicon esculentum) (Martin and Ort, 1985).
Several elements that contribute to this inhibition have been
identified and all may ultimately arise from the photosynthetic
production of oxygen radicals (Wise, 1995). An inhibition of
electron-transport capacity originating from damage to the reducing
side of PSII is well documented (e.g. Powles et al., 1983; Kee et al.,
1986; Percival et al., 1987) and, for moderately sensitive species such as maize, may be the major cause of impaired whole-plant photosynthesis after chilling. However, in the most severely chilling-sensitive species, such as domestic tomato, impaired reductive activation of the
stromal bisphosphatases appears to be the dominating factor limiting
carbon assimilation after chilling in the light (Sassenrath et al.,
1990).
Low temperature at night can also cause severe reductions in
CO2 fixation that persist on the day after the
chill, even after optimal growth temperatures have been restored. Like
the inhibition caused by chilling in the light, it is clear that the
primary loss of activity is caused by direct impairment at the
biochemical level, as opposed to interference with stomatal-mediated
leaf gas exchange (Martin et al., 1981). However, in the case of dark chilling, it has not been possible to assign the cause to specific reactions of photosynthesis. Thus, although the inhibition of net
photosynthesis by dark chilling in plants such as tomato and cucumber
can be quite large, the underlying causes are subtle and are likely to
involve disruption of the coordination among the component reactions of
photosynthesis rather than direct inhibition of the reactions
themselves. An intriguing effect of low temperature on the circadian
regulation of transcription (Martino-Catt and Ort, 1992) indicates that
the low-temperature-induced inhibition of photosynthesis in tomato may
result from the loss of coordination in the expression of critical
enzymes controlling photosynthetic metabolism.
In tomato, low-temperature treatment delays the progress of the
circadian clock regulating the transcription of certain nuclear-encoded genes, including cab (chlorophyll
a/b binding) and rca
(Rubisco activase) (Martino-Catt
and Ort, 1992). The clock stops for the duration of the chilling
treatment and resumes upon rewarming, but the affected rhythms are then
out of phase with the actual time of day. Notably, whereas
cab and rca gene expression are circadian
regulated in chilling-tolerant spinach, the rhythm is not affected in
this fashion by low-temperature treatment (Ort et al., 1989). However,
cab and rca are exceedingly abundant, stable proteins, and this
transitory mistiming in their synthesis in chilling-sensitive plants
does not lead to detectable changes in cellular protein level (Cooper
and Ort, 1988). Therefore, this mistiming cannot be expected to cause
the severe inhibition of net photosynthesis that is observed. Although
the chilling-sensitive aspect of circadian regulation is unknown, it is
an interesting possibility that low temperature could delay the timing
of circadian-regulated activity of certain enzymes, as we observed for
circadian-regulated gene transcription (Martino-Catt and Ort, 1992).
Such delays in circadian activity rhythms would likely have a negative
effect on photosynthetic performance if the affected enzymes were
critical control points of cellular carbon or nitrogen metabolism.
SPS (EC 2.4.1.14) is a central enzyme in photosynthetic metabolism
because it catalyzes a rate-limiting step in Suc biosynthesis. SPS is
subject to multiple levels of regulation, including regulation by the
allosteric effectors Glc-6-P and phosphate (Doehlert and Huber, 1984;
McMichael et al., 1993) and regulation by protein phosphorylation
(Huber et al., 1989). Our 1997 study showed that SPS activity exhibits
diurnal and circadian rhythms in tomato, which are the result of
corresponding oscillations in SPS protein phosphorylation state (Jones
and Ort, 1997). To our knowledge, this was the first report of a
circadian rhythm in SPS activity, although an endogenous ultradian
rhythm with a period of about 12 h had been reported in soybean
(Kerr et al., 1985). Our evidence indicates that the circadian rhythm
in tomato SPS phosphorylation state is the result of
circadian-regulated transcription of a protein phosphatase, possibly
the one that dephosphorylates and thereby activates SPS. Even a
transitory mistiming in transcription of this phosphatase gene as a
result of low-temperature treatment might potentiate a change in the
pattern of SPS activity. Because the capacity to use triose phosphate
can limit photosynthesis (Herold, 1980; Sharkey, 1990), it would be
anticipated that mistimed SPS activity as a result of low-temperature
treatment could contribute significantly to the chilling-induced
inhibition of photosynthesis.
NR (EC 1.6.6.1) activity exhibits a circadian rhythm in many plant
species (Lillo, 1984; Deng et al., 1990; Cheng et al., 1991; Pilgrim et
al., 1993). NR is a highly regulated cytosolic enzyme catalyzing the
first and rate-limiting step in the nitrate assimilation pathway,
reducing nitrate (NO3
) to
nitrite (NO2). The regulation
of NR activity is complex and can involve modulation of the enzyme
level through the regulation of synthesis and degradation (Solomonson
and Barber, 1990; Hoff et al., 1994; Kaiser and Huber, 1997). However,
immediately after a light-to-dark transition, rapid posttranslational
modifications of the enzyme are thought to dominate the regulation of
NR activity (Kaiser and Spill, 1991; MacKintosh, 1992; Kaiser and
Huber, 1994). Inactivation of NR in the dark is initiated by
phosphorylation of a specific seryl residue (Douglas et al., 1995;
Bachmann et al., 1996; Su et al., 1996) followed by the
Mg2+-dependent association of 14-3-3-type
inhibitor proteins with phospho-NR (Spill and Kaiser, 1994; Bachmann et
al., 1995; Glaab and Kaiser, 1995; MacKintosh et al., 1995; Lillo et
al., 1997). The similarities between the regulation of SPS and NR in
spinach (Huber et al., 1992a), especially the role of protein
dephosphorylation in enzyme activation and similar diurnal activity
dynamics, prompted us to investigate whether NR activity in tomato is
regulated by a circadian rhythm and, if so, whether this rhythm is
driven by changes in protein phosphorylation state, as we have shown
for SPS (Jones and Ort, 1997). We further investigated the effect of
low-temperature treatments on the timing of NR activity in tomato.
We have shown that, whereas the circadian rhythm in SPS activity in
tomato corresponds to oscillations in SPS phosphorylation state, the
circadian rhythm in NR activity is primarily the result of oscillations
in the amount of enzyme present in the leaves. We present data
demonstrating that low temperature delays the circadian rhythms in both
SPS and NR activity. Inhibitor treatments were used to investigate the
mechanism of the low-temperature shift, and the results indicate that
delayed activity involves low-temperature effects on the expression of
critical protein phosphatase(s). We believe that the low-temperature
inhibition of photosynthesis in chilling-sensitive plants is caused by
low-temperature-induced mistiming of the normal diurnal activity
pattern of key enzymes, thereby disrupting photosynthetic and cellular
metabolism.
| |
MATERIALS AND METHODS |
Plant Growth Conditions
Tomato (Lycopersicon esculentum Mill. cv Floramerica)
plants were grown from seed in growth chambers under a 14-h (26°C)
light/10-h (21°C) dark cycle at 75% RH, as described by Jones and
Ort (1997). Plants were fertilized twice weekly with a liquid formula
(12-31-14, Plant Marvel Laboratories, Chicago, IL) supplemented with 10 mM KNO3. All samples were taken from
young, fully expanded leaves of plants 21 to 28 d after planting.
Low-Temperature Treatments
Potted tomato plants were chilled at 4°C at 100% RH. During
chilling the pots were enclosed in an insulated box fitted with a fan,
which circulated warm air around the pots. This apparatus maintained
the soil temperature at approximately 15°C and completely prevented
the plants from wilting during treatment and rewarming.
SPS Assay
In vitro SPS activity was assayed colorimetrically by the anthrone
method to monitor the formation of Suc (and Suc-6-P) from Fru-6-P and
UDP-Glc (Huber et al., 1992b). Tomato extracts and assay conditions
were as described by Jones and Ort (1997).
NR Assay
In vivo NR activity was assayed using the leaf disc method of
Nicholas et al. (1976). Weighed leaf punches (approximately 200 mg)
were vacuum infiltrated in 10 mL of incubation buffer (0.1 M potassium phosphate [pH 7.5], 0.05 M
KNO3, 1% [v/v] propanol). Samples were
incubated in a shaking water bath for 30 min at 30°C in the dark.
After the incubation, 0.4 mL of the incubation buffer was diluted with
water to 4 mL. Two milliliters of 1% sulfanilic acid in 1.5 M HCl was added to the diluted incubation buffer, followed
by 2 mL of N-(1-naphthyl)ethylenediamine-HCl (200 mg/L). The
samples were then incubated for at least 20 min at room temperature to
allow full color development, after which the
A540 was recorded.
The measurement of in vitro NR activity using crude tissue extracts
followed a protocol that was modified from Kaiser and Brendle-Behnisch
(1991). Excised leaf samples were immediately frozen in liquid nitrogen
and stored at 
80°C until use. The samples were ground under liquid
nitrogen and then suspended in extraction buffer (1 g of tissue for
each 2 mL of buffer) consisting of 50 mM Mops-NaOH (pH
7.5), 10 mM MgCl2, 1 mM
EDTA, 5 mM DTT, and 0.1% Triton X-100. The homogenate was
subsequently desalted on Sephadex G-25 spin columns that had been
preequilibrated in 50 mM Mops-NaOH (pH 7.5) and 2 mM DTT. The desalted tissue extract (100 µL) was added to
900 µL of reaction solution containing 50 mM Mops-NaOH (pH 7.5), 10 mM KNO3, 0.1 mM NADH, and either 10 mM
MgCl2 or 5 mM EDTA. The samples were
incubated at 30°C for 3 min and the reaction stopped by the addition
of 100 µL of 50 mM zinc acetate. NO2 was quantified by standard
colorimetric reagent addition and the A540
was recorded (Lillo, 1983). The blanks were identical to the samples,
but were quenched with zinc acetate before the addition of the leaf
extract.
NR activity was calculated on a chlorophyll basis. The chlorophyll
concentration was assayed from the crude homogenates after an 80%
acetone extraction and calculated according to the method of Graan and
Ort (1984).
Inhibitor Treatments
Attached tomato leaves were lightly abraded with 400-grit Duralum
powder (Electro Minerals Corp., Niagara Falls, NY) and rinsed before
application of cordycepin (200-500 µg/mL), cycloheximide (200 µg/mL), staurosporine (100 µM), or okadaic acid (10 µM). All inhibitors were dissolved in 0.5% to 1% Tween
20. Okadaic acid and staurosporine were applied in 100-µL droplets to
the attached tomato leaves or the leaves were briefly submerged in the
cordycepin or cycloheximide solutions. The incubation times were as
described in the figure legends.
RNA Gel-Blot Hybridization
Total RNA (30 µg/lane) prepared from leaf samples was separated
by electrophoresis on agarose-formaldehyde gels and transferred to
nitrocellulose membranes. The membranes were hybridized overnight at
50°C with a NR probe prepared from a tobacco nia-2 cDNA
insert (Vaucheret et al., 1989) isolated from an Escherichia
coli plasmid and randomly labeled with
dCTP32. The membranes were washed three times for
5 min at 50°C in a solution of 150 mM NaCl, 15 mM sodium citrate, and 0.1% SDS followed by three 20-min
washes at 50°C in 30 mM NaCl, 3 mM sodium
citrate, and 0.1% SDS. A corresponding ethidium bromide-stained gel
was used to monitor the uniformity of total RNA loaded in each lane. The radioactivity bound to the membranes was quantified by 24 h of
exposure on a phosphor screen before analysis with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
| |
RESULTS |
The Circadian and Diurnal Rhythms of SPS Activity in Tomato Are
Delayed by Low-Temperature Treatment
SPS activity oscillates with a circadian rhythm in tomato when
plants are maintained under constant light (50 µmol quanta m2 s1) and temperature
conditions (Jones and Ort, 1997). The rhythm of SPS activity was
monitored after a 12-h low-temperature treatment (4°C, 50 µmol
quanta m2 s1) depicted
by the gray area in Figure 1. Samples
were taken after rewarming, and the results demonstrate that the
low-temperature treatment delayed the circadian rhythm in SPS activity
by 12 h, indicating that the endogenous clock controlling SPS
activity stopped for the duration of the chill and resumed upon
rewarming.
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| Figure 1.
Low-temperature treatment delays the circadian
rhythm controlling SPS activity. Plants were transferred to constant
low-light (50 µmol quanta m2 s1) and
temperature (26°C) conditions. The light and dark bars above the
figure represent day and subjective night periods, respectively. A
low-temperature treatment (4°C), represented by the shaded area in
the main body of the figure, was imposed for 12 h, between h = 29 and h = 41. After rewarming, samples were taken to determine
the effect of low temperature on the circadian rhythm in SPS activity.
Comparison of the reference circadian rhythm ( ) and the postchilling
oscillation ( ) demonstrates that the 12-h low-temperature treatment
delayed the oscillation in SPS activity by 12 h. Each point
represents the mean of two samples, and the experiment was repeated
several times with consistent results. Chl, Chlorophyll.
|
|
SPS activity also has a pronounced diurnal rhythm in tomato, with high
activity during the day and lower activity at night (Jones and Ort,
1997). Figure 2 shows that the SPS
diurnal rhythm was also delayed by low-temperature treatment. Dark
chilling treatment initiated at midday (h = 9), when SPS activity
was high, resulted in the maintenance of high activity upon rewarming,
even though the plants were rewarmed at a time of day when SPS activity
should be low (h = 22) (Fig. 2, top). Samples taken during the
dark recovery after the chilling episode showed that, although SPS
activity was initially high, the activity decreased to nighttime
levels during the next 8 h (Fig. 2, top).
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| Figure 2.
The effect of dark chilling treatments initiated
at different times in the diurnal cycle on SPS activity in tomato. The
white bar above the figure represents the light cycle, the black bar
represents the dark cycle, and the gray bar represents the continuation
of darkness during the normal light cycle (i.e. subjective day). Top,
The dark chilling treatment was initiated during the light cycle at
h = 9 and plants were rewarmed at h = 22. Data points were
normalized to the diurnal SPS activity profile at h = 24. Bottom,
The dark chilling treatment (4°C) was initiated during the night at
h = 17, plants were rewarmed (26°C) at h = 24, and samples
were then taken in the dark after rewarming (). Data were normalized
to the diurnal activity profile at h = 22. In both experiments,
the low-temperature treatment resulted in the maintenance of the
prechill SPS activity, showing that low-temperature treatment
delayed the normal rhythm in SPS activity. Chl, Chlorophyll.
|
|
Dark chilling treatment initiated during the night (h = 17), when
SPS activity was low, resulted in low activity upon rewarming, even
when plants were rewarmed at a time when SPS activity should be high
(h = 24; Fig. 2, bottom). However, after rewarming, SPS activity
remained low and did not continue the chill-delayed oscillation (Fig.
2, bottom). The reason that SPS activity did not recover as expected is
not certain but is likely the result of the extended dark period. We
have been unable to follow a circadian rhythm in SPS activity in
constant darkness because, after an initial oscillation during the
first subjective night, SPS activity decreased to low levels and
remained there (data not shown).
Low-Temperature Treatment Delays SPS Activity by Delaying the
Oscillation of the Protein Phosphorylation State
To determine if the inappropriately high SPS activity after the
chilling treatment (Fig. 2, top) might be caused by maintenance of the
prechilling phosphorylation state, we use the Ser/Thr kinase inhibitor
staurosporine, which is an effective inhibitor of SPS deactivation in
tomato (Jones and Ort, 1997). Staurosporine (100 µM) was
applied (Fig. 3) to lightly abraded,
attached tomato leaves immediately upon rewarming (h = 22) and
samples were harvested 4 h later (h = 26). The staurosporine
treatment prevented SPS deactivation after chilling, suggesting that
the maintenance of high SPS activity was caused by maintenance of the
prechilling dephosphorylated state of the enzyme. Cycloheximide (200 µg/mL), an effective inhibitor of cytoplasmic translation when
applied under these conditions (Jones and Ort, 1997), did not prevent the postchilling deactivation of SPS. This result showed that, although
SPS protein kinase activity is required for the postchilling deactivation of SPS, the kinase was already present and did not need to
be synthesized de novo. Therefore, the delay in the circadian rhythm of
SPS activity was the result of delayed changes in protein phosphorylation state, very possibly resulting from an effect of
chilling on the circadian rhythm controlling the transcription of a
protein phosphatase (perhaps SPS phosphatase) gene(s).
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| Figure 3.
Inhibitor studies show that SPS maintains the
prechilling phosphorylation state after chilling. Light/dark cycles are
represented by the shaded bars above the figure, as described in the
legend to Fig. 2. A dark chill was imposed between h = 9 and
h = 22, and immediately upon rewarming staurosporine (100 µM,  ) or cycloheximide (200 µg/mL,  ) was applied
to attached, abraded tomato leaves. The time of inhibitor application
is indicated ( ). After a 4-h dark incubation, samples were taken to
determine SPS activity. The staurosporine treatment maintained SPS in
the more active state, demonstrating that the prechilling,
dephosphorylated form of SPS was maintained throughout the
low-temperature treatment. Each point represents the mean of at least
three samples, and the SD values were less than 15% of the
mean values. Chl, Chlorophyll.
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The Circadian Pattern in NR Activity in Tomato Is Driven Primarily
by Changes in NR Expression
There was a robust circadian oscillation in NR activity in tomato
under constant light (450-500 µmol quanta m2
s1) and temperature (26°C) whether assayed
under in vivo (Fig. 4, top) or in vitro
(Fig. 4, bottom) conditions. This circadian profile is very similar to
that reported by Lillo (1984) for NR activity in barley.
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| Figure 4.
NR has a circadian pattern in transcript and
protein levels, which is responsible for an endogenous rhythm in NR
activity in tomato. The light and dark bars above the figure represent
day and subjective night periods, respectively. Top, NR activity ()
was assayed under in vivo conditions (see ``Materials and Methods'')
over a 3-d, constant light (450 µmol quanta m2
s1) and temperature (26°C) time course. The experiment
was repeated twice, and the results shown are representative. gfw,
Grams fresh weight. Bottom, NR activity was assayed
under in vitro conditions in the absence () and presence ( ) of
Mg2+. The absence of Mg2+ prevents the
Mg2+-dependent binding of 14-3-3-type inhibitor proteins to
phospho-NR, thereby revealing changes in NR activity that are caused by
changes in enzyme level (see text). Total leaf RNA was isolated from
tomato leaves under constant-light circadian conditions and probed with
a dCTP32-labeled tobacco nia-2 cDNA ().
The radioactivity was quantified on a phosphor imager. All experiments
were repeated at least three times and representative results are
shown. Chl, Chlorophyll.
|
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Light activation of NR activity can involve both changes in NR protein
level and changes in enzyme phosphorylation state (Solomonson and
Barber, 1990; Huber et al., 1992a; Crawford and Arst, 1993; Pilgrim et
al., 1993; Ramalho et al., 1995). We used the in vitro assay to resolve
changes in activity associated with the NR phosphorylation status and
subsequent Mg2+-dependent binding of 14-3-3-type
inhibitor proteins to phospho-NR (Spill and Kaiser, 1994; Bachmann et
al., 1995; Glaab and Kaiser, 1995; MacKintosh et al., 1995). Under
continuous-light conditions, in the absence of
Mg2+, NR activity was stimulated and still
circadian (Fig. 4, bottom). The fact that the
Mg2+-dependent inhibition of NR activity did not
increase (Fig. 4, bottom) during the subjective night, when NR activity
was lowest, is reasonably strong evidence that the circadian rhythm in
NR activity is not primarily dependent on changes in the enzyme's phosphorylation state.
Kaiser and Huber (1997) showed that, when phosphorylated, full
activation of spinach NR by the removal of Mg2+
can require as much as 30 min, which could obscure regulation of
activity by changes in NR phosphorylation. For tomato leaf samples
taken at the peak (32 h) or trough (24 h) of continuous-light circadian
NR activity, there was no evidence of slow activation in the presence
of EDTA (Fig. 5). Kaiser and Huber (1997)
also included the NR activators AMP and Pi during the incubation with EDTA, but we observed no added effect of these activators in our experiments (data not shown). The decline in NR activity over the
course of the incubation shown in Figure 5 was most likely caused by
artificial inactivation of the enzyme and was considerably more
pronounced in the absence of protease inhibitors or if the incubation
was performed at room temperature (data not shown). An indication of
slow activation of tomato NR upon removal of Mg2+
was observed in tomato leaves after 20 h of dark adaptation. The
extent of the slow activation would be very pronounced if corrected for
the time-dependent decline in activity seen in the continuous-light
samples. It should be noted that the chlorophyll content on a
fresh-weight basis was approximately 20% higher for tomato under
continuous light than after 20 h in the dark, such that maximum NR
activity on a fresh-weight basis was actually lower in dark-adapted
leaves. This difference in slow activation of NR between light- and
dark-adapted leaves is consistent with the report of Lillo et al.
(1997). Thus, unlike SPS (Jones and Ort, 1997), the dynamics of NR
phosphorylation do not play a dominant role in the circadian regulation
of this enzyme.
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| Figure 5.
Continuous-light circadian samples do not show
slow activation of NR in the presence of EDTA (i.e. in the absence of
Mg2+). Leaf samples were taken at the peak (32 h,  ) or
trough (24 h, ) of continuous-light circadian NR activity and after
20 h of dark adaptation (). The data plotted are the average of
three separate experiments.
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Figure 4 (bottom) further shows that the NR transcript level oscillated
in synchrony with the phase of NR circadian activity. Whereas this may
suggest circadian control of transcription, as we demonstrated
previously in tomato for both cab and rca
expression (Martino-Catt and Ort, 1992), there is evidence that
circadian oscillations of NR mRNA in Arabidopsis (Pilgrim et al., 1993) and maize (Redinbaugh et al., 1996) are controlled primarily by posttranscriptional events. Figure 6
shows that the circadian increase in NR activity that would normally
occur at 32 h was inhibited by pretreatment of the leaves with the
transcription inhibitor cordycepin (300 µg/mL) and the translation
inhibitor cycloheximide (200 µg/mL) whether activity was assayed in
the presence (data not shown) or absence (Fig. 6) of
Mg2+. As we observed for SPS (Jones and Ort,
1997), okadaic acid (10 µM), a potent inhibitor of types
1 and 2A protein phosphatase, also completely inhibited the circadian
increase in NR activity whether Mg2+ was present
or absent in the assay. Okadaic acid also significantly diminished the
circadian increase in NR mRNA, as it has been observed to do for the NR
mRNA increase after a dark-to-light transition in maize (Redinbaugh et
al., 1996).
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| Figure 6.
The circadian increase in NR activity () is
prevented by inhibitors of translation and transcription; the circadian
increase in NR activity and transcript level is prevented by inhibitors
of protein phosphatases. The light and dark bars above the figure
represent day and subjective night periods, respectively. Tomato leaves
were treated with okadaic acid (10 µM, and ),
cordycepin (200 µg/mL, ), or cycloheximide (200 µg/mL, ) at
the times indicated (). NR activity was assayed under in vitro
conditions. NR activity data are representative of at least four
separate samples, and were normalized to the continuous-light circadian
rhythm at h = 32. Total leaf RNA (, ) was probed with a
dCTP32-labeled tobacco nia-2 cDNA. NR
transcript data are the average of two separate samples, and were
normalized to the circadian rhythm at h = 55. Chl, Chlorophyll.
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Low-Temperature Treatment Delays the Circadian Rhythm in NR
Activity
The shaded area in Figure 7 depicts
a 6-h low-temperature treatment imposed during continuous-light
circadian conditions at a NR activity peak. Samples were taken after
rewarming and assayed in the absence of Mg2+. The
low-temperature treatment delayed the circadian rhythm in NR activity
by approximately 6 h, indicating that the endogenous clock
controlling NR activity stopped for the duration of the chill and
resumed upon rewarming. Assays performed in the presence of
Mg2+ showed an essentially identical response (at
50% lower NR activity) to the low-temperature treatment (data not
shown).
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| Figure 7.
Low-temperature treatment delays the circadian
rhythm controlling NR activity. The light and dark bars above the
figure represent day and subjective night periods, respectively. A
low-temperature treatment (4°C), represented by the shaded area in
the main body of the figure, was imposed for 6 h, between h = 38 and h = 44. After rewarming, NR activity was assayed under in
vitro conditions in the absence of Mg2+ (). Comparison
of the reference circadian rhythm () and the postchilling
oscillation () demonstrates that the 6-h low-temperature treatment
delayed the oscillation in NR activity without Mg2+ (i.e.
NR protein level). The experiment was repeated several times with
consistent results. Chl, Chlorophyll.
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|
Because chilling in conjunction with high light inhibits photosynthesis
in tomato by a different and more rapidly developing mechanism than
chilling in the dark (Sassenrath et al., 1990), we wanted to validate
the result shown in Figure 7 under dark conditions. Using the in vivo
assay, we observed a highly reproducible initial dark-circadian
increase in NR activity at h = 24, subjective dawn, but the
activity then decreased to very low levels (Fig. 8, bottom). Low-temperature treatment
(4°C) was imposed during the diurnal dark cycle, h = 14 to 24, and the plants were rewarmed in the dark (26°C). NR activity was
monitored after rewarming, and Figure 8 (bottom) reveals that the
low-temperature pretreatment delayed the dark-circadian increase in NR
activity just as it did in the light (Fig. 7). Comparing the control
dark-circadian oscillation with the chill-delayed oscillation, it is
clear that the circadian timing was offset by the 10-h duration of the
chill.
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| Figure 8.
Low temperature delays the circadian control of
the rise in in vivo NR activity that normally occurs before dawn. Top,
For these experiments, plants grown under diurnal conditions
(light/dark cycles are represented by the shaded bars above the figure,
as described in the legend to Fig. 2) were given extended darkness in
place of the normal dark-to-light transition at h = 24 (subjective
day is represented by the gray bar). NR activity () was assayed in
the dark during the night and the following subjective day. Okadaic
acid (10 µM, ) inhibited the circadian increase in NR
activity. The experiment was repeated three times, and the profile
shown is a representative result. Bottom, Plants were chilled at 4°C
during the diurnal night cycle, between h = 14 and h = 24, and rewarmed in the dark. Samples were taken after rewarming () and
demonstrate that the low-temperature treatment delayed the increase in
NR activity by 10 h, indicating that the circadian clock stopped
for the duration of the chill and resumed upon rewarming. During the
dark recovery phase, okadaic acid (10 µM, ) was
applied to attached tomato leaves (h = 32). Two hours later
(h = 34), in vivo NR activity was assayed from the
inhibitor-treated leaves and surfactant-treated controls. This
experiment was repeated three times with consistent results. gfw, Grams
fresh weight.
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Inhibitor treatments indicated that the low-temperature delay in the NR
circadian rhythm involved protein phosphatase activity (Fig. 8,
bottom). After chilling treatment, okadaic acid (10 µM) was applied to attached leaves at h = 31, when NR activity was low. Samples were harvested 2 h later, when NR activity was
expected to be high (h = 33). The okadaic acid treatment
completely abolished the chill-delayed increase in NR activity and the
normal diurnal increase in NR activity (Fig. 8, top). We obtained
qualitatively indistinguishable results compared with those presented
in Figure 8 using the in vitro NR assay in both the presence and the
absence of Mg2+ (data not shown).
| |
DISCUSSION |
Several plant genes have circadian oscillations in transcription,
including those for cab (Millar and Kay, 1991; Kellmann et al., 1993),
Fd (Bringloe et al., 1995), catalase (Zhong et al., 1994), and rca
(Martino-Catt and Ort, 1992). Frequently, however, there are not
corresponding oscillations in enzyme activity because of large, stable
protein pools. In fact, few proteins have been shown to have circadian
oscillations in enzyme activity; those that have include NR (Lillo,
1984), SPS (Jones and Ort, 1997), and PEP carboxylase (Carter et al.,
1991). Because circadian fluctuations are exhibited in many overt
higher-plant processes, including leaf movement (Darwin, 1880; Satter
and Galston, 1981), stomatal resistance (Martin and Meidner, 1971; Kerr
et al., 1985; Holmes and Klein, 1986), and net photosynthesis
(Chia-Looi and Cumming, 1972; Hennessey and Field, 1991), it seems
unavoidable that many more enzymes must exhibit a circadian component
to their regulation than the few so far identified. Circadian
fluctuations in free cytosolic calcium (Johnson et al., 1995) may also
play an important role in mediating the rhythms in these complex plant processes.
We demonstrated circadian regulation of SPS (Jones and Ort, 1997) and
NR activity (Fig. 4) in tomato. The circadian pattern of NR activity
under continuous-light conditions was driven primarily by changes in
the protein level, which mimicked oscillations in the NR transcript
level. That the percentage of inhibition by Mg2+
did not change in phase with the rhythm showed that, unlike those of
SPS, the dynamics of NR phosphorylation play no significant role in the
circadian regulation of this enzyme.
Although circadian control of SPS and NR activity responded similarly
to inhibitors that affect the phosphorylation status of proteins and to
inhibitors of translation and transcription, it is likely that they did
so for quite different reasons. In contrast to that in NR, the
circadian rhythm in SPS activity occurs in the absence of any
corresponding changes in SPS protein level, and corresponds to
circadian oscillations in the SPS phosphorylation state, which in turn
appears to be driven by a circadian pattern of SPS-phosphatase gene
transcription (Jones and Ort, 1997). Whereas the transcription
inhibitor cordycepin and the translation inhibitor cycloheximide
blocked the circadian increase in NR activity (Fig. 6), this was very
likely caused by direct effects of these inhibitors on NR expression.
Okadaic acid treatment inhibited the circadian increase in NR activity
even though the increase was clearly not caused by the
dephosphorylation of NR. Okadaic acid also inhibited the circadian
increase in NR mRNA accumulation (Fig. 6). It has been proposed that
separate protein phosphatases are involved in regulating NR translation
and transcript accumulation (Redinbaugh et al., 1996). Thus, the
transcriptional and/or translational components to the circadian
regulation of NR expression may be at multiple levels.
The circadian rhythm in SPS and NR activity in tomato was delayed by
exposure to low temperature (Figs. 1 and 7) in a manner analogous to
the chilling-induced delay in the circadian rhythm of cab
and rca transcription that we described earlier
(Martino-Catt and Ort, 1992). Perhaps more physiologically relevant,
the diurnal rhythm in SPS (Fig. 2) and NR (Fig. 8) activity was also
delayed by low-temperature treatments, so a cool night will result in mistiming of activity the next day.
Impaired photosynthesis as a result of mistimed SPS activity can be
reconciled with current understanding of the regulation of
photosynthetic carbon metabolism. Insufficient SPS activity would be
expected to limit the rate of photosynthesis (Herold, 1980; Sharkey,
1990; Micallef et al., 1995) by causing an accumulation of triose
phosphate in the chloroplast at the expense of free phosphate (Sharkey,
1990). It is proposed that the limited availability of free phosphate
in the chloroplast would reduce the ATP-generating capacity of the
chloroplast ATP synthase, thereby ultimately leading to the inhibition
of photosynthesis (Sharkey, 1990).
Genetically transformed plants with modified levels of enzymes in Suc
biosynthesis have been examined for altered photosynthesis. Overexpressed maize SPS results in substantial increases in SPS activity, as well as an increased capacity for Suc synthesis in transformed tomato (Worrell et al., 1991; Galtier et al., 1993; Micallef et al., 1995). Under standard atmospheric
CO2 conditions there was no statistically
significant increase in photosynthesis; however, when grown under
doubled atmospheric CO2, the maize
SPS-transformed lines supported a 20% greater rate of photosynthesis
(Galtier et al., 1993; Micallef et al., 1995). Increasing atmospheric
CO2 concentrations increases the likelihood that
plants will experience feedback inhibition because this condition
allows photosynthesis to outpace end-product (triose phosphate)
utilization. The higher photosynthetic rates in the SPS-overexpressing
plants indicates that increased Suc synthesis can alleviate some of
this feedback inhibition. Similar results are seen in Flaveria
linearis transformants with altered levels of cytosolic
Fru-1,6-bisphosphatase (Micallef et al., 1996), another key regulatory
enzyme in Suc biosynthesis. Photosynthesis at atmospheric levels of
CO2 is not significantly affected in these
mutants (either over- or underexpressing cytosolic Fru-1,6-bisphosphatase) compared with controls; however, at elevated CO2 levels the predicted effects based on
feedback inhibition are seen. The lines overexpressing cytosolic
Fru-1,6-bisphosphatase showed increased levels of photosynthesis,
whereas those underexpressing this enzyme were unable to take full
advantage of the elevated CO2.
Although the mistiming of SPS alone might be expected to lead to the
inhibition of photosynthesis in tomato, our results suggest that the
underlying basis for the inhibition of photosynthesis in
chilling-sensitive plants is the cumulative result of the mistiming of
numerous circadian-controlled enzyme activities. Here we show that the
circadian rhythm in both SPS and NR activity was arrested in tomato by
chilling and that an effect of low temperature on critical protein
phosphatases was involved in each case. For both enzymes, it appears
that it is a protein phosphatase that is under circadian regulation,
and that this circadian rhythm is delayed by low-temperature treatment.
These results imply that other, as-yet-unidentified circadian-regulated
enzyme activities, particularly those regulated by protein
phosphorylation, may be affected by low-temperature treatment as well.
Because the circadian rhythm in SPS activity seems to be controlled by
the transcription of SPS phosphatase, and the SPS activity rhythm is
delayed by low-temperature treatment, it is a sensible hypothesis that
the mechanism for the low-temperature delay in SPS activity is a delay
in the circadian rhythm in SPS-phosphatase transcription. However, the
fact that NR behaves identically to chilling exposure even though
phosphorylation of NR does not contribute to the circadian regulation
of activity indicates that low temperature may act at an earlier,
common step in the regulation pathway of these enzymes. Because okadaic
acid-sensitive protein phosphatase activity is critical to the
circadian regulation of both of these enzymes, it would be a plausible
chill-sensitive common link that could have far-reaching effects on the
photosynthetic metabolism in chill-sensitive plants.
| |
FOOTNOTES |
1
This work was supported in part by the U.S.
Department of Agriculture National Research Initiative Competitive
Grants Program (grant no. 91-37100-6620 to D.R.O.) and by an
Integrative Photosynthesis Research Training Grant from the Department
of Energy (no. DEFGO2-92ER20095), funded under the program for
Collaborative Research in Plant Biology.
2
Present address: MJ Research, Inc., 136 Coolidge Avenue, Watertown, MA 02172.
*
Corresponding author; e-mail d-ort{at}uiuc.edu; fax
1-217-244-0656.
Received March 20, 1998;
accepted June 8, 1998.
| |
ABBREVIATIONS |
Abbreviations:
NR, nitrate reductase.
SPS, Suc phosphate
synthase.
| |
ACKNOWLEDGMENTS |
We thank Erin Grant and Sonja Kemmis for their able assistance
in conducting a portion of the SPS and NR assays, and Jim Harper's laboratory for assistance with the in vivo NR assays.
| |
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Top
Abstract
Introduction
