Plant Physiol. (1999) 120: 887-896
Photosynthesis and Carbon Partitioning in Transgenic Tobacco
Plants Deficient in Leaf Cytosolic Pyruvate Kinase1
Bernard Grodzinski*,
Jirong Jiao,
Vicki L. Knowles, and
William C. Plaxton
Department of Plant Agriculture, University of Guelph, Guelph,
Ontario, Canada N1G 2W1 (B.G., J.J.); and Departments of Biology
(V.L.K., W.C.P.) and Biochemistry (W.C.P.), Queen's University,
Kingston, Ontario, Canada K7L 3N6
 |
ABSTRACT |
Whole-plant diurnal C exchange
analysis provided a noninvasive estimation of daily net C gain in
transgenic tobacco (Nicotiana tabacum L.) plants
deficient in leaf cytosolic pyruvate kinase (PKc
).
PKc plants cultivated under a low light intensity (100 µmol m
2 s1) were previously shown to
exhibit markedly reduced root growth, as well as delayed shoot and
flower development when compared with plants having wild-type levels of
PKc (PKc+). PKc and
PKc+ source leaves showed a similar net C gain,
photosynthesis over a range of light intensities, and a capacity to
export newly fixed CO2 during photosynthesis.
However, during growth under low light the nighttime, export of
previously fixed 14CO2 by fully expanded
PKc leaves was 40% lower, whereas concurrent respiratory
14CO2 evolution was 40% higher than that of
PKc+ leaves. This provides a rationale for the reduced root
growth of the PKc plants grown at low irradiance. Leaf
photosynthetic and export characteristics in PKc and
PKc+ plants raised in a greenhouse during winter months resembled those of plants grown in chambers at low irradiance. The data
suggest that PKc in source leaves has a critical role in
regulating nighttime respiration particularly when the available pool
of photoassimilates for export and leaf respiratory processes are low.
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INTRODUCTION |
PK catalyzes the synthesis of pyruvate and ATP from PEP and ADP
and is believed to be a major control point of plant and nonplant glycolysis (Plaxton, 1996
). The enzyme has been demonstrated to be
significantly displaced from equilibrium in vivo and has pronounced regulatory properties in vitro. Plant PK exists as cytosolic and plastidic isozymes (PKc and
PKp, respectively), which differ substantially in
their molecular and kinetic/regulatory properties.
PKc plays an important role in generating the
precursor pyruvate for various biosynthetic pathways and mitochondrial
respiration. The biosynthetic role of cytosolic glycolysis is central
in actively growing autotrophic tissue (Plaxton, 1996), in which a
significant proportion of the C that enters the glycolytic pathway is
incorporated into numerous compounds such as amino acids, nucleic
acids, fatty acids, and secondary metabolites. The exact contribution
that these enzymatic steps provide in source leaves and in developing
sink tissues remains unclear.
PKc deficiency in nonplant species causes serious
detrimental effects. However, our earlier studies revealed that
transgenic tobacco plants (Nicotiana tabacum L.) deficient
in leaf PKc (PKc) grew
from seed to seed, demonstrating the remarkable flexibility of plant
PEP metabolism (Gottlob-McHugh et al., 1992; Knowles et al., 1998).
Plant cells can use a variety of alternative metabolic routes to
directly or indirectly circumvent the reaction catalyzed by
PKc. These could include the action of PEP
phosphatase or the combined action of PEP carboxylase, malate
dehydrogenase, and NAD-malic enzyme (Plaxton, 1996). It is also
possible that the elevated levels of PEP observed in the
PKc leaves (Gottlob-McHugh et al., 1992)
results in an increased flux of glycolytic C from the cytosol to the
chloroplast where the PEP may be metabolized by
PKp. Elimination of leaf
PKc can alter C metabolism and growth when total
C supply is limited by growing the plants at reduced PPFD. Knowles et
al. (1998) showed that, when grown at low PPFD, the
PKc tobacco exhibited a delayed shoot and
flower development, as well as a striking reduction in root growth.
Since the lack of PKc and the resulting altered
glycolytic activity appeared to be confined to the leaves, we decided
to further investigate the role of the leaves as sources of reduced
C.
We recently described a procedure for evaluating immediate export rates
during photosynthesis so that we could test and differentiate between
the effect of environmental challenges, such as leaf warming, on the
ability of the sources leaves to fix CO2 and to
export the reduced C products (Jiao and Grodzinski, 1996; Leonardos et al., 1996). These protocols were modified to study the effect of
short- and long-term CO2 enrichment on
photosynthesis and export rates in source leaves of a number of
C3 and C4 species (Jiao and
Grodzinski, 1996, 1998; Grodzinski et al., 1998). The aim of the
present study was to use these procedures to assess whole-plant gas
exchange, photosynthesis, respiration, and export in intact, attached
source leaves of two independent homozygous PKc
tobacco lines. We report that, when both PKc
lines were cultivated under low irradiance, nighttime export of
recently fixed CO2 was reduced, whereas
concurrent respiration of 14C assimilates was
enhanced. These findings provide a rationale for the reduced root
development of the PKc plants.
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MATERIALS AND METHODS |
Plant Materials
Two transgenic tobacco (Nicotiana tabacum L.) lines
that specifically lacked PKc in their leaves were
obtained because of the trans-inactivation phenomenon known
as cosuppression (Gottlob-McHugh et al., 1992). Selfing of each parent
line resulted in two PKc progeny lines, 14-1 and 15-7, in which the cosuppression was relatively stable, and a
PKc+ line, 18-7, which contained wild-type levels of PKc (Knowles et al., 1998). The seeds obtained
from the selfing of 14-1 and 15-7 (designated
PKc1 and PKc2,
respectively) plus the wild type and 18-7 (PKc+1
and PKc+2, respectively) were used in the present
study. Seeds were germinated and grown in PROMIX-BX (Les
Tourbières Premier LTÉE, Rivière du Loup, QC, Canada) in 20-cm pots in growth chambers at the University of Guelph. A 16-h
photoperiod was maintained at 22°C ± 1°C during which the PPFD (400-700 nm) was 500 µmol m2
s1 (moderate light) or 100 µmol
m2 s1 (low light).
During the 8-h dark period the temperature was 18°C ± 1°C.
Plants were fertilized biweekly with a nutrient solution, as described
previously (Knowles et al., 1998).
In other experiments plants were grown during winter months (i.e.
between November and March) in a greenhouse at the University of Guelph
(latitude approximately 43.5°N). Solar-generated PPFD varied from 50 µmol m2 s1 on
overcast days to more than 1000 µmol m2
s1 on sunny days. Artificial lighting was
supplied by 1000-W Sylvania metal halide lamps throughout the 16-h
photoperiod and maintained a minimum daytime PPFD of about 130 µmol
m2 s1 at the plant
level. Temperatures in the experimental greenhouse compartments were
typically 24°C ± 1°C/18°C ± 1°C, day (16 h)/night (8 h).
PK Assay and Immunoblot Analyses
All measurements were made on the most recently fully expanded
leaves, which were harvested, frozen in liquid
N2, and stored at 80°C until used. Enzyme
extracts were prepared from leaves, as described previously
(Gottlob-McHugh et al., 1992). PK activity was assayed
spectrophotometrically at 25°C, as described by Plaxton (1989), and
was corrected for PEP phosphatase activity by omitting ADP from the
reaction mixture. One unit of PK activity is defined as the amount of
enzyme resulting in the utilization of 1 µmol PEP
min1. Activity values represent the means of
quadruplicate determinations conducted with three separate extracts and
were reproducible to within ±10% SE. Protein
concentration was determined by the modified Bradford assay (Bollag and
Edelstein, 1991) using bovine 
-globulin as the standard. Extracts
were electrophoresed on 7.5% (w/v) SDS-polyacrylamide minigels and
electroblotted onto a PVDF membrane. Immunoblotting was performed using
affinity-purified anti-castor endosperm PKcIgG, and antigenic polypeptides were detected using an
alkaline-phosphatase-conjugated secondary antibody, as described
previously (Plaxton, 1989). Immunological specificities were confirmed
by conducting immunoblots in which rabbit preimmune serum was
substituted for the anti-PKcIgG.
Whole-Plant NCER and Daily C Gain
The growth rate of the plants was measured noninvasively by
determining the whole-plant NCER using whole-plant gas-exchange chambers, as described previously (Dutton et al., 1988). Measurements were made at 24°C ± 1°C/18°C ± 1°C, day (16 h)/night (8 h), and ambient CO2 (35 Pa) and
O2 (21 KPa). Plants grown under the moderate lighting regime were measured at 500 µmol m2
s1 PPFD at the level of the top leaf, whereas
the plants grown under low light were measured at 100 µmol
m2 s1 PPFD. One plant
per chamber was used for the measurements. Because four chambers were
run concurrently using a central IR gas-analysis system,
CO2 exchange measurements were made at 6- to
8-min intervals during a typical 50-h test (Leonardos et al., 1994).
Six replications were conducted and based on the average diurnal gas
exchange measurements; whole-plant daily C gain was calculated for each
line under both PPFD levels.
Leaf Net CO2 Exchange Rates
Plants were illuminated with Sylvania metal halide lamps (1000 W),
which could provide a maximum of about 1800 µmol
m2 s1 PPFD at the leaf
level. The light response curves for photosynthesis were derived by
using a series of neutral screens to reduce PPFD. The leaf gas-exchange
rates of the most recently expanded leaves of 8- to 9-week-old plants
were measured using an open-flow system described previously (Jiao and
Grodzinski, 1996). Both leaf and air temperature in the plant chamber
were maintained at 24°C ± 1°C. The inlet gas contained 35 Pa
CO2 and 21 KPa O2.
Chlorophyll content was determined as described by Wintermans and de
Mot (1965).
Export and Storage of 14C Assimilates during
Photosynthesis
Export of newly fixed 14C assimilates during
steady-state photosynthesis was estimated as described previously (Jiao
and Grodzinski, 1996). Plants were illuminated with Sylvania metal
halide lamps (1000 W), which provided about 1200 µmol
m2 s1 PPFD at the leaf
level. A GM detector was mounted in the lower half of the leaf cuvette
to continuously monitor radioactivity (i.e. 14C
accumulation) in the source leaves. To establish the time required to
reach an equilibrium between the
14CO2 of known specific
activity and the 14C-labeled Suc pool, leaves
were first fed with 14CO2
from 30 to 120 min and the labeled products were analyzed. The fed leaf
was extracted with boiling 80% ethanol:water (v/v), and the major
14C-labeled assimilates were analyzed as
described elsewhere (Jiao and Grodzinski, 1996). During feeding periods
net 14CO2 assimilation and
14C accumulation rates of attached intact leaves
were monitored continuously in a noninvasive manner using an IR gas and
a GM detector, respectively. The measurement of the export rate of the
newly fixed 14CO2 during
steady-state photosynthesis was calculated as the difference between
the rate of 14CO2
assimilation and the retention of 14C assimilates
90 to 120 min after 14CO2
feeding began. As reported below, isotopic equilibrium between the
specific activity of the 14C-Suc in the leaf and
that of the 14CO2 in the
gas stream was established during this period. The CO2 and O2 levels were 35 Pa and 21 KPa, respectively. Whole- plant and leaf temperatures were
24°C ± 1°C and the RH was approximately 70%.
Leaf Dark Respiration and Nighttime Export of
14C-Labeled Assimilates
The respiration and export of 14C
assimilates during the dark period that followed the
14CO2 feeding were
determined by trapping the
14CO2 released and
continuing to monitor the level of 14C in the
source leaf with the GM detectors mounted in the leaf cuvettes (Jiao
and Grodzinski, 1998). After the
14CO2 was supplied for
2 h and the rates of photosynthesis and concurrent export were
determined, the lamps were extinguished and the leaves were supplied
with a gas stream lacking
14CO2. The
CO2 and O2 levels were 35 Pa and 21 KPa during the 14-h chase period in the dark. The export of
14C in the dark was corrected for loss of
14CO2 due to respiration,
which was determined by trapping the gas in 20% (w/v) KOH and
measuring radioactivity by liquid-scintillation counting (Leonardos et
al., 1996).
| |
RESULTS |
PK Activity and Immunoblot Analysis
PK activity assays and immunoblotting using anti-castor endosperm
PKcIgG were used to assess the relative
abundance of PKc in extracts prepared from leaves
of the same tobacco plants used in the physiological studies described
below. The PK activities of fully expanded PKc+
and PKc leaves harvested from plants grown under moderate or low light are reported in Figure
1. Extractable PK activity of the
PKc leaves was reduced by 70% to 85% relative to the PKc+ controls. Two to three separate
extracts of the PKc+ and
PKc leaves were analyzed by immunoblotting, and
representative results are shown in Figure 1. Immunoblots of
PKc+ leaf extracts revealed an intense
immunoreactive polypeptide at 57 kD, which corresponds to subunits of
tobacco leaf PKc (Knowles et al., 1998). By
contrast, antigenic staining of PKc on
immunoblots of PKc leaf extracts was either
very faint (PKc1) or undetectable.
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| Figure 1.
Immunological detection and activities of
PKc in extracts prepared from leaves of PKc+
and PKc tobacco plants grown under moderate ("M"
lanes) or low ("L" lanes) light intensities. Crude extracts were
electrophoresed on 7.5% (w/v) SDS-polyacrylamide minigels (15 µg of
protein per lane) and transferred to a PVDF membrane. Immunoblotting
was performed using affinity-purified anti-castor endosperm
PKcIgG (Plaxton, 1989). PK activities represent the means
of quadruplicate determinations conducted with three separate extracts
and were reproducible to within ±10% SE. FW, Fresh
weight.
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Plant Growth, Leaf Area, and Chlorophyll Content
As reported previously (Knowles et al., 1998), the
PKc lines had a slower rate of development than
did the two PKc+ lines. Therefore, plants that
were at an identical developmental stage were compared. The data
reported here are from the stage at which the first flower bud was
visible. Total leaf area per plant of the four lines grown under 500 µmol m2 s1 PPFD
(moderate light) were similar (Table I).
However, when plants were raised under 100 µmol
m2 s1 PPFD (low light),
the total leaf area of the PKcl was less than that of the PKc+ controls.
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Table I.
Total leaf area and chlorophyll content at flowering
stage of tobacco plants cultivated under moderate or low light in
growth chambers
Each value represents the mean ± SE obtained with at
least six leaves on six different plants. Significant differences among
PKc+ and PKc lines by Student's t
test (P < 0.05) are indicated by superscript letters (a, b, c).
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When leaf chlorophyll content of the PKc and
PKc+ lines were compared at a single light
condition, the leaves of the PKc lines had more
chlorophyll than did the leaves of the PKc+
plants (Table I). Under moderate light leaves of each line had a
greater chlorophyll content than leaves of that same line had when
grown under low light (Table I).
Whole-Plant NCER and Daily C Gain
Figure 2 shows whole-plant NCER and
daily net C gain of each tobacco line grown and measured at 500 or 100 µmol m2 s1 PPFD.
Within each panel it is clear that photosynthesis at moderate light was
greater than that at low light. However, whole-plant photosynthesis of
the four lines was similar when compared at the same PPFD level (Fig.
2, A-D). The photosynthesis rates for PKc1 and
PKc2 plants grown and measured at the moderate
light intensity were 5.8 and 6.1 µmol C fixed
m2 s1, respectively.
Whole-plant dark respiration rates ranged from 1.2 µmol C released
m2 s1 in
PKc+1 to 1.6 µmol C released
m2 s1 in
PKc2. At the lower PPFD level, plant
photosynthesis rates were one-third that of those at the moderate PPFD
level, averaging 1.9 and 2.1 µmol C fixed m2
s1 for the PKc2 (Fig.
2D) and PKc1 lines, respectively (Fig. 2C). Dark respiration rates of plants grown under low light were also less
than those of plants grown under moderate light. For example, in plants
acclimatized to 100 µmol m2
s1 PPFD, the average rates of dark respiration
during the 8-h night period were approximately 0.56 and 0.66 µmol C
released m2 s1 for the
PKc+1 (Fig. 2A) and PKc2
lines (Fig. 2D), respectively.
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| Figure 2.
Whole-plant NCERs and net C gain during a 16-h
photoperiod and an 8-h dark period in tobacco: PKc+1 (A and
E); PKc+2 (B and F); PKc1 (C and G); and
PKc2 (D and H). NCER was measured when the first flower
bud appeared. Plants were both grown and measured at a PPFD of 500 (solid lines) or 100 (dashed lines) µmol m2
s1. The CO2 and O2 levels were 35 Pa and 21 KPa, respectively. Day and night temperatures during the
analysis were 22°C and 18°C, respectively. The data are the means
of at least four replications.
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At 500 µmol m2 s1
PPFD the daily C gain of all four tobacco lines was approximately 300 µmol C gained m2, which was approximately
3-fold that measured at 100 µmol m2
s1 PPFD (Fig. 2, E-H). The data confirm that
at the whole-plant level there was little difference in daily C gain
per leaf area among the four tobacco lines.
Leaf Photosynthesis at Varying Irradiances
When intact attached source leaves were tested individually, net
CO2 fixation rates of PKc
plants were similar to those of the PKc+ plants
grown at the same light level (Fig. 3).
All plants grown at the moderate PPFD exhibited higher rates of leaf
photosynthesis (Fig. 3A) than did plants grown at low PPFD (Fig. 3B).
The PPFD required to saturate leaf photosynthesis was greater in plants acclimatized to the higher PPFD. In all of the plants grown under moderate and low light, the PPFD required to saturate photosynthesis was approximately 900 and 700 µmol m2
s1, respectively (Fig. 3).
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| Figure 3.
Leaf photosynthetic light-response curves of
PKc+ and PKc tobacco plants. Net
photosynthesis was measured when the first flower bud appeared on
plants grown in growth chambers under 500 (A) or 100 (B) µmol
m2 s1 PPFD. Photosynthesis was measured at
25°C ± 1°C. The CO2 and O2 levels
were 35 Pa and 21 KPa, respectively. Each value represents the
mean ± SE obtained with at least six leaves on six
different plants.
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Establishing Conditions for Estimating Export Rates during
Photosynthesis
The 14C-labeling patterns in intact attached
source leaves from PKc and
PKc+ plants are compared in Figure
4. Leaves of each line that had been
grown at low or moderate PPFD were tested at similar stages of plant
development at a PPFD that saturates photosynthesis. For ease of
reporting the only results shown here are for leaves of the
PKc+1 line (Fig. 4, A, C, and E) and the
PKc1 line (Fig. 4, B, D, and F). During the
120-min feed period total
14CO2 fixation increased
linearly (Fig. 4, A and B). The net photosynthetic rates were lower in
plants grown at 100 µmol m2
s1 PPFD (refer to Figs. 3 and
5), but net CO2
fixation rates were also constant during the 2-h feed. As illustrated
in Figure 4, A and B, the rates of total 14C
retention in the source leaves were higher during the 1st h of the feed
and reflects the fact that the label was being randomized among
different intermediate pools before the export pools reached isotopic
equilibrium with 14CO2 in
the gas stream.
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| Figure 4.
14C-Labeling patterns of intact,
attached source leaves of PKc+1 (A, C, and E) and
PKc1 (B, D, and F) tobacco plants. A and B, Total
14CO2 fixation, 14C retention, and
14C export of leaves of PKc+1 (A) and
PKc1 (B) plants grown at 500 µmol m2
s1 PPFD. Measurements were made during a 2-h
14CO2 feed at 35 Pa CO2, 25°C,
and 1000 µmol m2 s1 PPFD. Total C
fixation (dashed line) was calculated from IR gas analyzer data. The
14C retention was monitored noninvasively with the GM
detector (solid line) and invasively ( ) following ethanol
extraction. Export during steady-state 14CO2
feeding (fine dotted line) was estimated as the difference between
total fixation and 14C retention in the leaf. C and D,
Accumulation of 14C-Suc in the source leaves of plants
grown under low and moderate PPFD (i.e. 100 and 500 µmol
m2 s1 PPFD), respectively, but analyzed at
a saturating PPFD for photosynthesis. E and F, Time course for changes
in specific activities of 14C-Suc in the source leaves of
plants grown under low and moderate PPFD levels, respectively. Each
point represents the mean value (±SE) obtained with four
leaves of four different plants.
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| Figure 5.
Leaf photosynthesis (entire bar in A-C),
concurrent export rate (solid bar in A-C), and relative export flux
during photosynthesis (D-F) of tobacco plants with wild-type levels of
PKc (PKc+1 and PKc+2) and two lines
deficient in leaf PKc (PKc1 and
PKc2) grown under 500 (A) or 100 (B) µmol
m2 s1 PPFD in growth chambers or in the
greenhouse (C). Photosynthesis and concurrent export rates were
measured when the first flower bud was visible. Photosynthesis and
export were calculated from data obtained 90 to 120 min after
14CO2 was first supplied at a leaf temperature
of 25°C and at 1000 µmol m2 s1 PPFD, as
described in the legend to Figure 4 and in ``Materials and Methods''.
A to C, 14C partitioning into the total ethanol-insoluble
(Ins) fraction (i.e. starch), into the ethanol-soluble fraction (Sol),
and into that exported (Exp) from the leaf at the end of a 120-min feed
period. G to I, 14C partitioning into the total sugar
(Sug), organic acid (Org), and amino acid (Ami) fraction at the end of
a 120-min feed period. The height of each bar represents the mean of
measurements of at least four expanded leaflets of four different
plants.
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A major labeled product accumulating in all leaves during the 2-h feed
was 14C-Suc (Fig. 4, C and D). Photosynthesis was
greater and more 14C-Suc accumulated in leaves of
plants that had been grown under moderate light (Fig. 4, C and D, solid
line) than in those grown under low light (Fig. 4, C and D, dotted
line). Specific activities of 14C-Suc increased
rapidly in the leaves of the PKc+ plants during the 1st h of feed (Fig. 4E). The specific activities of
14C-Suc of plants grown under low light increased
similarly. However, the specific activity of
14C-Suc was lower in the
PKc leaves (Fig. 4F) in the
PKc+ leaves (Fig. 4E). The specific activity of
14C-Suc in PKc leaves was
slightly higher in the plants grown under low light. Nevertheless,
taken together the data clearly show that in all cases the specific
activity of the 14C-Suc was unchanged after 90 min (Fig. 4, E and F). Export rates reported below (Fig. 5) were
calculated from 14C retention and photosynthesis
rates obtained between 90 and 120 min of the feed when it was assumed
that the 14C-Suc was leaving the leaf as readily
as it was being synthesized from newly fixed
14CO2.
Estimates of Export Rates during Photosynthesis and 14C
Partitioning during the Feed
The photosynthesis rates of plants grown under moderate light
ranged from 14.5 to 16 µmol C fixed m2
s1 (Figs. 3 and 5A). All lines grown under low
light exhibited significantly lower leaf photosynthesis rates than
plants grown under moderate light (Figs. 3 and 5B). The photosynthesis
rates of plants grown in greenhouses during winter months were similar
to those raised in growth chambers at the lower PPFD level (Fig. 5, B
and C). Approximately 55% to 65% of the 14C
assimilated was exported immediately in plants cultivated in growth
chambers under either 500 (Fig. 5D) or 100 µmol
m2 s1 PPFD (Fig. 5E).
Only 45% to 50% was exported immediately in the plants grown in the
greenhouse (Fig. 5F). However, the immediate export rates of
14C-labeled photosynthate from leaves of both
PKc lines were statistically similar to those
of the PKc+ lines regardless of the growth
conditions (Fig. 5, D-F).
When leaves grown at the same light level were compared, there was no
difference in the partitioning of label into starch and sugars. There
appeared to be more label accumulated in starch in the plants growing
in the greenhouse (Fig. 5, C and F), which reflected the fact that the
immediate export rates were slightly lower in these plants. The
patterns of 14C distribution among the
ethanol-soluble pools (i.e. sugars, organic acids, and amino
acids) were similar in leaves of the PKc and PKc+ lines (Fig. 5, G-I). For, example based on
Student's t tests (not shown), there was no overall
difference in the amount of C accumulated in
organic acids in leaves of PKc and
PKc+ plants at the end of the 2-h feed period. In
all instances, 14C sugars accounted for the
largest pool (i.e. 60%-75% of the ethanol-soluble C products). The major observations were that
plants grown at 500 µmol m2
s1 PPFD had elevated photosynthesis rates and
stored 35% to 40% more 14C than did plants
grown at 100 µmol m2
s1 PPFD (Fig. 5, G and H). Previously fixed
14CO2 was primarily in the
form of 14C-Suc and
14C-starch at the end of the feed periods (Figs.
4, C and D, and 5, A-C, G-I). The fate of the
14C products that accumulated during the 2-h
labeling period was investigated further.
Leaf Dark Respiration and Nighttime Export of Labeled
Assimilates
Figure 6 shows examples of traces
obtained from GM detectors, which monitored the accumulation of
14C in leaves during the 2-h feeding period and
the disappearance of label during the subsequent 14-h chase in the
dark. Export and dark respiratory losses were variable during the
chase. Although it was not determined whether the rapid decrease in
radioactivity in the leaves at the beginning of the dark period was due
to increased export or respiration, the overall pattern was similar in
PKc+ leaves (Fig. 6, A and B) and
PKc (Fig. 6, C and D). During growth under
moderate light, dark export of 14C (Fig.
7A) and respiratory
14CO2 evolution (Fig. 7D)
were similar in leaves of all four lines. However, it is notable that
during growth under low light the rates of
14C-labeled photosynthate export in the dark were
about 40% lower (Fig. 7B) and respiratory
14CO2 evolution were 40%
greater (Fig. 7E) in the PKc leaves as compared
with the PKc+ controls. Similarly, during winter
months when natural PPFD levels are quite variable, leaves of
PKc plants raised in the greenhouse also
exported less 14C (Fig. 7C) and released more
14CO2 at night than did
leaves of the PKc+ controls (Fig. 7F).
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| Figure 6.
Effects of PKc deficiency in source
leaves of plants grown under low or moderate PPFD on the pattern of
14C accumulation during the 2-h feed in the light and the
subsequent disappearance of radioactivity during a 14-h chase period in
the dark, as monitored noninvasively with a GM detector. During the
dark chase period, CO2 and leaf temperature were maintained
at 35 Pa and 25°C, respectively. Each trace represents a typical
analysis profile obtained from a single leaf. The experiment was
repeated four times with the expanded leaves of four different
plants.
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| Figure 7.
Nighttime export (A-C) and respiration (D-F) of
previously fixed 14CO2 in leaves of
PKc+ and PKc plants grown in growth chambers
under moderate (A and D) or low (D and E) light or in a greenhouse
during winter months (C and F). Dark export was corrected for
respiration, which was determined by estimating the
14CO2 released from the source leaves during
the chase period. The height of each value represents the mean ± SE of measurements of four expanded leaves of four
different plants. Significant differences among PKc+ and
PKc lines by Student's t test (P < 0.05) are indicated by superscript, italic letters (a to
g) adjacent to the histobars.
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DISCUSSION |
Leaves of the tobacco plants used in the present investigation
were either deficient in PKc or they possessed
wild-type levels of the enzyme (Fig. 1). PK activities of
PKc leaves were only 15% to 30% of those of
PKc+ leaves. These reductions in PK activity are
similar to those previously reported for leaves of the
PKc plants (Gottlob-McHugh et al., 1992;
Knowles et al., 1998). Furthermore, antigenic staining of
PKc was either absent or barely detectable on
immunoblots of PKc leaf extracts (Fig. 1).
Residual PK activity of PKc leaves has been
attributed to PKp (Gottlob-McHugh et al., 1992).
The transgenic tobacco deficient in leaf PKc
exhibited a marked reduction in root growth, which became much more
pronounced during growth under low light (Knowles et al., 1998). The
objective of this study was to investigate the possibility that primary photoassimilate export from the source leaves was somehow modified by
the deficiency of leaf PKc.
The reduction in leaf PKc (Fig. 1) did not affect
whole-plant net C gain (Fig. 2), leaf photosynthesis (Figs. 3-5),
14C partitioning in the leaf (Fig. 5G), or
capacity of the leaf to export newly fixed CO2 in
the light or export stored 14C assimilates during
a subsequent nighttime chase when the plants were grown at 500 µmol
m2 s1 (Fig. 7A). These
results are consistent with those of Gottlob-McHugh et al. (1992), who
demonstrated that leaves of the PKc
transformants grown under 400 µmol m2
s1 PPFD exhibited normal rates of
photosynthetic O2 evolution (and respiratory
O2 consumption). There were no differences
between plant groups in the capacity of their leaves to assimilate
CO2 over a range of PPFD levels, including the
two PPFD levels at which the plants were grown. Similarly, the rates of
export of newly fixed C during a
14CO2 feeding were
identical. These data indicate that in source leaves the
PKc is not an absolute prerequisite for (a)
CO2 fixation, (b) metabolism of
14C photoassimilates, or (c) phloem loading and
export during photosynthesis (i.e. in the light). The major difference
between the PKc and the
PKc+ plants, which would account for the delayed
development and reduced root growth of the PKc
lines grown under low light, was the increased nighttime allocation of
previously fixed C to respiration versus export (Fig. 7, B and E). The
reduction in nighttime export could have a cumulative, negative
influence on sink development.
At the time of appearance of the first flower bud, this organ functions
as a strong sink for 14C assimilates (B. Grodzinski and J. Jiao, unpublished data). At this stage, about 55% to
60% of the 14CO2 being
fixed was being exported immediately (Fig. 5, D and E). Also, about
40% to 45% of the 14C photoassimilates remained
in the leaf (Fig. 5, D and E). The C retained
in the leaves of all plants was available for further metabolism and
export during dark periods. The plants acclimatized to 500 µmol
m2 s1 PPFD fixed more
14CO2 and therefore
retained more 14C at the end of the feed than did
the plants cultivated under 100 µmol m2
s1 PPFD. Thus, there are two factors affecting
mobilization and respiration rates during the dark chase period in the
PKc lines, which must be considered in these
experiments. The first is that these plants were clearly deficient in
PKc (Fig. 1). The second equally important
consideration is that photosynthesis rates were lower and the pool
sizes of 14C intermediates were correspondingly
lower in all plants acclimatized to 100 µmol
m2 s1 versus plants
acclimatized to 500 µmol m2
s1 PPFD. It appears that the
PKc step of cytosolic glycolysis can effectively
be bypassed by alternative enzyme(s) if there are sufficient reserves
of intermediates to drive other reaction sequences. In plants grown
under low light there may be a higher, proportional respiratory cost
associated with the translocation of 14C
intermediates at night. The enzyme (or enzymes) being used to bypass
PKc in the PKc less
leaves may not regulate glycolytic flux in support of dark respiration
as effectively as PKc. Thus, the dark respiration
of photoassimilates might be elevated, which could drain the pool of
available Suc for phloem loading/translocation to sinks (hence,
reducing export).
In cotton plants export in the dark was highly correlated with
carbohydrate levels (i.e. starch) at the end of the photoperiod and
with dark respiration rates (Hendrix and Grange, 1991). To some extent,
14CO2 respiratory losses
may reflect dark respiration rates in the leaves, which might be
providing energy to sustain export in the dark. Nighttime export
requires additional energy than in the light that can only be derived
from stored photoassimilates (Giaquinta, 1972; Côté et al.,
1992; Geiger and Servaites, 1994; Jiao and Grodzinski, 1998). Bouma et
al. (1995) calculated that as much as one-third of the C stored in
starch could be lost through dark respiration in support of the energy
requirements for photoassimilate mobilization and export. However, in
the PKc lines an increased respiration rate
(i.e. 14CO2 production) may
not reflect an increased rate of ATP production via oxidative
phosphorylation. There may in fact have been a reduction in ATP
production via oxidative phosphorylation. In the absence of normal
levels of PKc some or all of the "enhanced"
respiratory CO2 loss might occur via the
nonphosphorylating (cyanide resistant) pathway of mitochondrial
electron transport (involving the alternate oxidase).
PKc regulation may be very important to the
partitioning of electrons between phosphorylating and
nonphosphorylating pathways of mitochondrial electron transport, since
pyruvate is apparently the key activator of the alternative
oxidase/CN-resistant pathway of mitochondrial respiration (Day et al.,
1994). Thus, in PKc leaves a depletion of
stored assimilates via a bypass pathway coupled with a reduced supply
of energy via oxidative phosphorylation for loading and translocation
processes may effectively jeopardize the opportunity for the
PKc leaf tissue to mobilize carbohydrate reserves at night.
The PKc leaves had higher rates of
14CO2 loss and exported
less 14C during the dark than did
PKc+ plants, but only during cultivation under
low light when the total amount of 14C
assimilates produced was reduced. The plants were at a similar stage of
development and the relative rates of export during photosynthesis (i.e. export expressed as a percentage of photosynthesis) of these leaves were similar (Fig. 5, D and E). A primary difference between the
PKc plants grown at low versus moderate light
was the amount of 14CO2
initially fixed during the feed (Fig. 5, A, B, G, and H). Further
studies are required to distinguish how PKc and
PKc bypass enzymes are regulated when plants are
cultivated under very low irradiances. Our previous study revealed
that, to compensate for the physical deficiency of leaf
PKc, the PKc leaves did
not up-regulate the activities the PKc bypass
enzymes PEP phosphatase or PEP carboxylase (Knowles et al., 1998).
Thus, fine regulation of preexisting PKc bypass
enzymes may allow the PKc leaves to partially
cope with PKc deficiency. Further studies are
required to assess whether this is the case and, if so, whether it
might be achieved by the "constitutive" phosphorylation of the leaf
PEP carboxylase into its phosphorylated, more active (i.e. less malate
inhibited) state.
Leaves of PKc+ and PKc
plants raised in a greenhouse during winter months demonstrated
photosynthesis (Figs. 5C), dark export, and respiration characteristics
(Figs. 7, C and F) that were similar to those of plants grown in
chambers under low light (Figs. 5B and 7, B and E, respectively).
Growing the test plants in a greenhouse during our winter months
represents a different light-limited growth condition. The plants
cultivated in the greenhouse were subject to wide fluctuations in PPFD
during the photoperiod. Most plants in nature are exposed to varying
levels of PPFD during their development. Diurnal transitions are
obvious, but cloud cover, sun flecking, and mutual shading are also
commonplace events influencing leaf metabolism and function (Boardman,
1977). Our results suggest that the presence of
PKc and the operation of a "normal"
glycolytic pathway are important in the production of energy needed for
the mobilization of photoassimilates, when energy derived in the light
from photosynthesis is not (directly) available, or when the levels of
carbohydrates destined for export are too low to drive Suc synthesis,
loading, and translocation processes required to meet sink demand.
In this study total nighttime export rates were not measured in all of
the leaves in the canopy. Rather, the disappearance of label following
a 2-h period of 14CO2
labeling during which a steady rate of photosynthesis was maintained.
In sugar beet, following longer labeling periods (e.g. 6 h), it
was shown that during the first 2 to 3 h in the dark the source of
C for export was from the Suc pool that accumulated during the light
period and that the concentration of Suc was an important factor
controlling the rate of translocation during the subsequent dark period
(Geiger and Batey, 1967). Thereafter, starch was mobilized to form Suc
for translocation. In tobacco C-Suc accumulated
during the feed period (Fig. 4, C and D) and likely served as a source
of exported 14C during the nighttime chase (Fig.
6). In the tobacco leaves acclimatized to growth at either the low or
the moderate light, the fastest rate of disappearance of previously
fixed 14C occurred during the first hours of the
dark period (Figs. 6 and 7). In this study nighttime export would be
underestimated in all of the lines. A reduced export rate in the
PKc lines during the dark chase period means
that the actual reduction in translocation of photoassimilates among
these plants was probably much greater than that reported here.
Taken together, our results underscore the problems of raising
transgenic plants in different growth conditions and/or relying solely
on data derived during growth under one light condition. To our
knowledge, this is the first study in which the expression in leaves of
a glycolytic enzyme was altered and photosynthate export rates were
measured both in the light and in the dark. Selected modification of
other reactions associated more directly with C transport and
phloem-loading processes do alter translocation (Lerchl et al.,
1995; Geigenberger et al., 1996; Hattenbach et al., 1997;
Hausler et al., 1998). A major difference noted in the transgenic
plants deficient in leaf PKc was the reciprocal effect on C export and dark respiration in low-light-grown plants that
were not assimilating CO2 at a fast rate. This
was not observed when the PKc plants were grown
at a moderate light level. Further insight into the role of
PKc and glycolysis in maintaining homeostasis in
source leaves may be provided by challenging transgenic plants with
growth conditions in which nutrients, as well as light conditions, are
varied.
| |
FOOTNOTES |
1
This work was supported by research and
equipment grants to B.G. and W.C.P. from the Natural Sciences and
Engineering Research Council of Canada, and grants to B.G. from the
Ontario Ministry of Agriculture and Food and Rural Affairs, Flowers
Canada Ontario Ltd., the Cecil Delworth Foundation, and the Centre for
Research in Environmental and Space Technology.
*
Corresponding author; e-mail bgrodzinski{at}evbhort.uoguelph.ca;
fax 1-519-767-0755.
Received December 14, 1998;
accepted April 15, 1999.
| |
ABBREVIATIONS |
Abbreviations:
GM, Geiger-Müller.
NCER, net carbon
exchange rate.
PK, pyruvate kinasePKc and
PKp,.
cytosolic PK and plastid PK, respectively.
PKc and PKc+, tobacco plants that are and are
not deficient in leaf PKc, respectively.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge Mr. George Lin for his technical
assistance during the analysis of 14C assimilates
and Mr. Luke Lairson for his help preparing figures for this
manuscript.
| |
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Abstract
Introduction