Plant Physiol. (1999) 119: 1125-1136
Decrease in Phosphoribulokinase Activity by Antisense RNA in
Transgenic Tobacco. Relationship between Photosynthesis, Growth, and
Allocation at Different Nitrogen Levels1
Fiona M. Banks,
Simon P. Driscoll,
Martin A.J. Parry,
David W. Lawlor,
Jacqui S. Knight,
John C. Gray, and
Matthew J. Paul*
Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden,
Hertfordshire AL5 2JQ, United Kingdom (F.M.B., S.P.D., M.A.J.P.,
D.W.L., M.J.P.); and Department of Plant Sciences, University of
Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom (J.S.K,
J.C.G.)
 |
ABSTRACT |
To study the direct effects of
photosynthesis on allocation of biomass by altering photosynthesis
without altering leaf N or nitrate content, phosphoribulokinase (PRK)
activity was decreased in transgenic tobacco (Nicotiana
tabacum L.) with an inverted tobacco PRK cDNA and plants were
grown at different N levels (0.4 and 5 mM
NH4NO3). The activation state of PRK increased
as the amount of enzyme was decreased genetically at both levels of N. At high N a 94% decrease in PRK activity had only a small effect (20%) on photosynthesis and growth. At low N a 94% decrease in PRK
activity had a greater effect on leaf photosynthesis (decreased by up
to 50%) and whole-plant photosynthesis (decreased by up to 35%) than
at high N. These plants were up to 35% smaller than plants with higher
PRK activities because they had less structural dry matter and less
starch, which was decreased by 3- to 4-fold, but still accumulated to
24% to 31% of dry weight; young leaves contained more starch than
older leaves in older plants. Leaves had a higher ion and water
content, and specific leaf area was higher, but allocation between
shoot and root was unaltered. In conclusion, low N in addition to a
94% decrease in PRK by antisense reduces the activity of PRK
sufficient to diminish photosynthesis, which limits biomass production
under conditions normally considered sink limited.
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INTRODUCTION |
The relationship between photosynthesis and growth is a complex
one, with growth rate not being well correlated with the rate of
photosynthesis on a leaf-area basis (Poorter et al., 1990
). This is
because growth also depends on the investment of biomass in growing
sinks and investment in leaf area (Chapin et al., 1990; Poorter and
Remkes, 1990). There have been many thorough investigations of the
relationship between photosynthesis, growth, environment, and genotype,
but they have not provided definitive information on the relationship
between photosynthesis and growth. This is because of the complex
interaction between factors, so it has not been easy to decide whether
changes in sink growth result from changes in photosynthesis or vice
versa. The elucidation of the relationship between photosynthesis and
growth requires specific changes in photosynthesis or sink growth
independent of other changes. Genetic manipulation of enzymes involved
in photosynthesis and/or sink processes provides a means to achieve this. The use of transgenic plants with altered amounts of Rubisco revolutionized the analysis of photosynthesis and its interaction with
the whole plant (Quick et al., 1991b; Stitt and Schulze, 1994).
However, because Rubisco constitutes such a large proportion of the
protein in a leaf (up to 40%; Woodrow and Berry, 1988), a decrease in
amounts of Rubisco substantially disrupts the N balance of the plant,
making it difficult to establish direct links between photosynthesis
and growth and allocation than if there were a more specific alteration
in the rate of photosynthesis. What is required is genetic modification
of a photosynthetic enzyme that accounts for a fraction of the N
content in the leaf.
PRK is one such enzyme, accounting for less than 1% of the protein in
a leaf (Evans, 1989). Tobacco (Nicotiana tabacum L.) plants
were transformed with an inverted cDNA encoding tobacco PRK (Knight and
Gray, 1994). First, it was determined that the effect of this
modification on photosynthesis under standard growth-chamber conditions
was minimal until there was a large decrease in PRK activity (greater
than 85%), and even then the effects were small (Paul et al., 1995).
Later, down-regulation of PSII activity and electron transport (Habash
et al., 1996) and the possible contribution of differences in amounts
of tight-binding inhibitors to differences in activation of Rubisco in
these plants (Paul et al., 1996) were documented.
Here we use these plants to study the direct effects of photosynthesis
on allocation of biomass. To test the hypothesis that a genetic
decrease in photosynthesis would have less effect on growth under
strongly sink-limited conditions, we grew the plants at low-N
(sink-limited) and high-N levels. Measurements were carried out on four
lines of plants with activities of PRK between 6% and 100% of the
wild type. To demonstrate predictable effects of antisense during the
course of plant development and with N nutrition, measurements of a
number of parameters were made on leaves and plants of different ages.
These data are plotted against developmental stage (number of leaves
per plant). Where measurements are made at just one stage of
development, the data are plotted against PRK activity.
| |
MATERIALS AND METHODS |
Plant Material
Tobacco (Nicotiana tabacum L.) plants were transformed
by Agrobacterium tumefaciens-mediated leaf-disc
transformation. Tobacco PRK cDNA was subcloned into the binary vector
pROK8, a derivative of pBIN19, with a tobacco rbcS promoter
and nos terminator. Of the 11 homozygous lines originally
characterized (Paul et al., 1995) four were used in the experiments.
Line 4 was transformed in the same way as the others, but with the PRK
sequence omitted from the construct, which gave wild-type activities of
PRK. These plants served as the control plants. Line 1 had activities
of PRK that were on average 6% of the wild type; line 2, 54%; and line 3, 73%. These activity differences between the lines were maintained at both high and low N, in leaves of different ages, and in
plants of different ages (Fig. 1, A-D). Plants were
T2 progeny of selfed T1
obtained from the independent transformants T0.
During the experiments we took care to ensure that plants did not shade
each other during growth. Seeds were sown on filter paper and
transferred to pots of Bedfordshire silver sand when seedlings were
10-d-old (Joseph Arnold, Leighton Buzzard, UK). Then they were
irrigated to field capacity with nutrient medium containing either
(final concentrations) 5 mM
NH4NO3 (high N) or 0.4 mM
NH4NO3 (low N) and 3 mM
KH2PO4, 1 mM MgSO4, 1 mM CaSO4, 20 µM Fe-EDTA, 50 µM
KCl, 25 µM
H3BO3, 1.5 µM MnSO4, 2 µM ZnSO4, 0.5 µM CuSO4, and 0.5 µM
H2MoO4. Aluminum foil was
placed on the surface of the sand when the plants were young to prevent
algal growth. Plants were cultivated in a controlled-environment
chamber providing 330 µmol photons m
2
s1, a 14-h photoperiod, and a constant
temperature of 25°C with 70% RH. Samples were taken from plants
between the 8-leaf and the 23-leaf stage. For plants grown at high N,
this was over a period of 28 d, and for plants at low N, this was over
a period of 69 d. The 8-leaf stage was 55 d from sowing for
both sets of plants. Samples for PRK, Rubisco, starch, and gas-exchange
measurements were taken from leaves of different ages. These were the
most recently fully expanded leaf (mid-leaf), which was then used as a
reference point for old leaves, taken two leaves below this one, and
young leaves, taken two leaves above it. These leaves represented the
most photosynthetically active part of the plant. Samples were taken
7 h into the photoperiod. At the 22-leaf stage, the element
content and enzyme activation states were also determined.
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| Figure 1.
Maximum catalytic activities of PRK in lines 1 (A), 2 (B), 3 (C), and 4 (D) grown at high N ( ,  ,  ) and low N
( ,  ,  ). Points are means of four replicates taken during the
middle of the photoperiod from the most recently fully expanded leaves
(, ), two leaves below these leaves (old leaves; , ), and
two leaves above these leaves (young leaves; , ). Error bars
represent the maximum SE of the different-aged leaves.
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Measurement of PRK and Rubisco Activities
To determine PRK and Rubisco activities, leaf samples were freeze
clamped using tongs with aluminum clamps precooled in liquid N2. PRK and Rubisco were extracted from 3 cm2 of leaf in 1 mL of 100 mM
Hepes-KOH, pH 8.0, 10 mM MgCl2, 0.4 mM EDTA, 0.1% Triton X-100, and 15 mM
2-mercaptoethanol in a Potter homogenizer at 4°C. For the assay of
PRK, aliquots were diluted 20-fold in extraction buffer and assayed
immediately at 25°C by coupling the formation of ADP to the oxidation
of NADH using pyruvate kinase and lactate dehydrogenase in the presence
of 20 mM DTT to ensure full activation of PRK (Kagawa,
1982). Initial activities of PRK were measured as described by Leegood
(1990), except that the reaction was allowed to proceed for just
30 s in the absence of 20 mM DTT. After 30 s the
reaction was quenched with 5% perchloric acid and the amount of ADP
was measured as in the assay for maximum activity. Rubisco was assayed
in 100 mM Bicine, pH 8.2, containing 20 mM
MgCl2, 100 mM
NaH14CO3 (0.5 µCi
µmol1), and 33 mM
ribulose-1,5-bisphosphate. Initial activities were determined from 20 µL of undiluted extract and assayed for 1 min before quenching with
100 µL of 10 N formic acid. Maximum Rubisco activity was
determined after preincubation of extract in assay buffer minus
ribulose-1,5-bisphosphate for 3 min. The assay was then started with
ribulose-1,5-bisphosphate and quenched with 10 N formic
acid after 1 min. The incorporation of 14C label
was determined by scintillation counting.
Measurement of Photosynthesis
Rates of net photosynthesis of individual leaves were measured in
the laboratory under the conditions in which the plants had been grown
(330 µmol photons m2
s1, 350 µmol CO2
mol1, 25°C, 70% RH, and 1 kPa vapor pressure
deficit). Measurements were carried out using a six-chamber
open-circuit gas-exchange system with automatic data handling. The
partial pressure of CO2 was controlled by a gas
blender (Signal Instruments Co., Croydon, UK) and measured with an IR
gas analyzer (Mark 3, ADC, Hoddesdon, UK). The humidity of the air
before and after passage over the leaf was determined with capacitance
sensors (Vaisala, Helsinki, Finland). All measurements were made on
10-cm2 areas of the most recently fully expanded
leaves attached to 6-week-old plants in leaf chambers with forced
ventilation. The flow rate to each leaf chamber was 9 cm3 s1. The
CO2 concentration within the leaf was calculated
as described by von Caemmerer and Farquhar (1981). Leaves were allowed
to equilibrate to conditions within the chambers for 30 min before
measurement. Rates of photosynthesis of whole plants were measured in a
Perspex chamber (70 cm high, 40 cm wide, and 40 cm deep) connected to the gas-exchange system described above, with all conditions the same.
The flow rate to the chamber was 194 cm3
s1 and RH in the chamber was 50%. Whole plants
were allowed to equilibrate to the conditions for 90 min
before measurement. The gas exchange of the roots in sand was also
determined and taken into account in the final calculation of
whole-plant photosynthesis.
Determination of Growth Parameters
Leaf area was measured using an automated planimeter (Delta-T
Devices, Ltd., Burwell, Cambridge, UK). Dry weight was measured after
plant material was dried in an oven at 70°C. SLA was calculated as
the amount of leaf area generated per unit dry weight invested in the
leaf, and LAR was calculated as the amount of leaf area per unit total
dry weight.
Measurements of Carbohydrates
Discs were cut with a cork borer and extracted in 100 mM imidazole-HCl buffer, pH 6.9, and an aliquot was
immediately added to 80% ethanol at 70°C for 20 min. Samples were
then kept at 
20°C until analysis. Glc, Fru, and Suc were measured
through the reduction of NADP by Glc-6-P dehydrogenase after the
sequential addition of hexokinase, phosphoglucose isomerase, and
invertase (Jones et al., 1977). The method was adapted for use on an
ELISA plate, using reduced volumes and a microplate reader to measure
absorbance changes. Starch was measured after breakdown of starch to
Glc in the insoluble pellet using 
-amylase and amyloglucosidase
(Sonnewald et al., 1991) after extraction of soluble components from
leaf discs. The Glc was then measured as described above.
Measurement of Elements and Nitrate
Leaves were dried and ground to a fine powder in a mill. C and N
contents were measured by combustion in a LECO C, N, and S analyzer
(model CNS-2000, LECO Instruments, Ltd., Stockport, Cheshire, UK).
Macronutrients (S, K, Mg, Na, P, and Ca) were extracted from dried leaf
material by digestion with a nitric/perchloric acid mixture and
quantified by inductively coupled plasma emission spectroscopy (Maxim,
Applied Research Laboratories, Ecublens, Switzerland). Nitrate was
extracted from dried leaf material in boiling water and converted to
nitrite with nitrate reductase, and the nitrite was quantified with
sulfanilic acid and N-(1-naphthyl)ethylenediamine dihydrochloride (Greiss reagent). A550 was
measured on a microplate reader (model MR5000, Dynatech Laboratories,
West Sussex, UK) (Misko et al., 1993; Verdon et al., 1995).
| |
RESULTS |
Activities of PRK and Rubisco
The activity of PRK was affected in a uniform way by antisense in
the four transgenic lines under low and high N and in leaves and plants
of different ages (Fig. 1, A-D).
Activity of PRK in line 1 was 6% of wild-type PRK activity
irrespective of plant or leaf age or nutrition; line 2, 54%; and line
3, 73%. Low N compared with high N decreased activities of PRK
between 2- and 3-fold in the most recently fully expanded and young
leaves and by 4- to 5-fold in older leaves. The effects of leaf age on
PRK activity were more pronounced at low N than at high N, with older leaves of low-N plants containing one-half of the activity of younger
leaves. At high N the effects of leaf age on PRK activity were
small. There was a small in-crease and then a decrease in PRK activity
with plant age in plants grown at high N, but little effect in plants
grown at low N.
Activities of Rubisco were also affected similarly in all lines by N
nutrition and by leaf and plant age (Fig.
2, A-D). Low N compared with high N
decreased Rubisco activities by about 3-fold. The effects of leaf age
on Rubisco activities at high N were more pronounced than the effects
of leaf age on PRK activities, with older leaves containing activities
up to one-third lower than younger leaves. The effect of leaf age at
low N was similar to the effect at high N and somewhat less pronounced
than the effect of leaf age on PRK activities at low N. There was a
small increase and then a decrease in Rubisco activity with plant age
in plants grown at high N, and little effect of plant age on Rubisco
activity at low N.
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| Figure 2.
Maximum catalytic activities of Rubisco in lines 1 (A), 2 (B), 3 (C), and 4 (D) grown at high N (, , ) and low N
(, , ). Points are means of four replicates from the most
recently fully expanded leaves (, ), two leaves below these
leaves (old leaves; , ), and two leaves above these leaves (young
leaves; , ). Error bars represent the maximum SE of
the different-aged leaves.
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|
The activation states of PRK increased in response to a decrease in the
amounts of PRK (P 
0.02; Fig. 3A).
The activation state of Rubisco also increased when PRK was decreased
genetically (P 0.001; Fig. 3B), except at low N, at which
Rubisco was fully active at all PRK activities.
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| Figure 3.
Activation state (initial activity as a percentage
of maximum activity) of PRK (A) and Rubisco (B) in leaves of plants
grown at high N (, , ) and low N (, , ). Line 1 (,
), line 3 (, ), and line 4 (, ). Each point represents a
measurement on a different plant of each line from the most recently
fully expanded leaves at the 22-leaf stage. Statistically significant
differences in PRK activation were recorded between line 1 and lines 3 and 4 at high and low N (P 0.02). Statistically significant
differences in Rubisco activation were recorded between line 1 and
lines 3 and 4 at high N (P 0.001).
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CO2 Assimilation
A decrease in PRK activity had an effect on
CO2 assimilation only in line 1 (Figs.
4, A-D, and
5). At high N the rates of CO2 assimilation of individual leaves and plants
of line 1 were 20% lower than those of the other lines when measured
under the growing conditions (Figs. 4, A-D, and 5A). However, at low N
the difference in CO2 assimilation between line 1 and the other lines was much greater and was greatest in older leaves,
in which rates of photosynthesis in line 1 were one-half of the rates
in the other lines (Fig. 4, A-D). In younger leaves photosynthesis in line 1 was about 60% of that in the other lines. Measured at the whole
plant, photosynthesis per plant of line 1 was 65% that of the other
lines at low N at the end of the experiment, the difference having
increased as the plants aged (Fig. 5B). At low N net
CO2 uptake actually decreased per plant beyond
the 14-leaf stage because of a large number of photosynthetically
inactive older leaves.
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| Figure 4.
Leaf photosynthesis in lines 1 (A), 2 (B), 3 (C),
and 4 (D) grown at high N (, , ) and low N (, , ).
Points are means of four replicates from the most recently fully
expanded leaves (, ), two leaves below these leaves (old leaves;
, ), and two leaves above these leaves (young leaves; , ).
Error bars represent the maximum SE of the different-aged
leaves. .
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| Figure 5.
Whole-plant photosynthesis in lines 1 (, ),
2 (, ), 3 ( ,  ), and 4 (, ) grown at high N (, ,
, ) and low N (, , , ). Error bars represent the
maximum SE of lines 2, 3, and 4, and the SE of
line 1.
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Growth and Allocation
Low N compared with high N produced smaller plants, which took
69 d from the 8-leaf stage to reach the 23-leaf stage, compared with 28 d at high N. Dry weight at the 23-leaf stage was 2-fold lower at low N compared with high N. The effect of a decrease in PRK
activity on growth and allocation at high N was minimal (Fig.
6, A, C, E, and G). At high N, plants of
line 1 were 10% smaller than plants of the other lines and there was
no consistent effect on the shoot-to-root ratio, SLA, and LAR. At low
N, however, stronger effects on growth of line 1 compared with the
other lines were observed (Fig. 6, B, D, F, and H). Plants with the
lowest PRK activities were 30% to 35% smaller by the end of the
experiment than the other lines (P 0.05; Fig. 6B), a difference
that became clear at the 10-leaf stage. This was because of smaller
shoots and roots, yet the shoot-to-root ratio was unaltered (Fig. 6D). From the 10-leaf stage, the SLA and the LAR of line 1 at low N were up
to 50% higher than in the other lines (Fig. 6, F and H) and almost
prevented the normal decrease of these parameters because of low N
(Fig. 6, E-H).
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| Figure 6.
Growth parameters of plants grown at high N (,
, , ) and low N (, , , ) in lines 1 (, ), 2 (, ), 3 (, ), and 4 (, ). Whole-plant dry weight (A
and B), shoot-to-root ratio (C and D), SLA (leaf area per unit leaf dry
weight; E and F), and LAR (leaf area per whole-plant dry weight; G and
H). Error bars represent the maximum SE of all of the lines
except for whole-plant dry weight, SLA, and LAR at low N, for which the
SE of line 1 is also shown.
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To explain these differences in dry weight and in investment in leaf
area, we measured the water, nutrient, and carbohydrate contents of the
leaves.
Water and Element Content of Leaves
Growth at low N compared with high N resulted in a lower
relative water content and lower amounts of K, Mg, and S as a
proportion of dry weight (Fig. 7, A, C,
D, and F). There was little effect of N nutrition on Ca and Na, and
amounts of P were slightly higher in line 1 at low N compared with high
N (Fig. 7, B, E, and G). There were no clear effects of PRK activity on
relative water content and inorganic ion content of plants grown at
high N (Fig. 7, A-G). At low N, however, there was a clear effect of
PRK activity on these parameters. The relative water content of leaves
of plants with the lowest activities of PRK grown at low N was 5%
higher than in plants with higher PRK activities (P 0.02; Fig.
7A). The inorganic element content of these leaves (Ca, K, Mg, S, and P, but not Na) was also higher (P 0.01) when expressed per unit dry weight (Fig. 7, B-G). The effects were most pronounced on K and P,
which were 2-fold higher in line 1 than in the other lines. Differences
persisted when data were expressed per unit leaf water (P 0.01)
or per unit structural dry weight (starch subtracted from dry weight;
P 0.05; data not presented in these forms).
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| Figure 7.
Water and element content in leaves of plants
grown at high N (, , , ) and low N (, , , ) in
lines 1 (, ), 2 (, ), 3 (, ), and 4 (, ).
Relative water content (A), Ca (B), K (C), Mg (D), Na (E), S (F), and P
(G) in lines 1 (, ), 2 (, ), 3 (, ), and 4 (,
). Each point represents a measurement on a different plant of each
line from the most recently fully expanded leaves at the 22-leaf stage.
Statistically significant differences in relative water content in
plants grown at low N were recorded between line 1 and lines 2, 3, and
4 (P 0.02) and for all elements except Na of plants grown at
low N between line 1 and lines 2, 3, and 4 (P 0.01).
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N, Nitrate, and C Content of Leaves
The antisense decrease in PRK activity had no effect on leaf N or
nitrate content, although low N significantly decreased by up to
10-fold the amounts of both (Fig. 8, A
and B). Amounts of C were decreased in line 1 compared with the other
lines at both N supplies, but the effect was more clear at low N (Fig. 8C). The C:N ratio was decreased by low PRK activity at low N (P 0.01) but was unaffected at high N (Fig. 8D).
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| Figure 8.
Amounts of N (A), nitrate (B), and C (C), and C:N
ratio (D), in leaves of plants grown at high N (, , , ) and
low N (, , , ) in lines 1 (, ), 2 (, ), 3 (,
), and 4 (, ). Each point represents a measurement on a
different plant of each line from the most recently fully expanded
leaves at the 22-leaf stage. Statistically significant differences in
the C content of plants grown at high N were recorded between lines 1 and 3 (P 0.01) and between line 1 and lines 2, 3, and 4 at low
N (P 0.001). Statistically significant differences in the C:N
ratio of plants grown at low N were recorded between line 1 and lines
2, 3, and 4 (P 0.01).
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Carbohydrate Content
There were only small effects of N nutrition and PRK activity on
the soluble carbohydrate content of leaves (data not presented). However, the effects of N and PRK activity on starch content were more
apparent (Fig. 9, A-D). At high N starch
was 20% to 50% lower in line 1 than in the other lines. There were no
large effects of leaf or plant age on starch content at high N. Low N
compared with high N increased starch content by up to 10-fold, and the amounts of starch were more markedly affected by PRK activity than at
high N. In line 1 starch was 3- to 4-fold lower than in the other
lines. Furthermore, the normal pattern of starch accumulation (highest
in older leaves and lowest in young leaves) was reversed in line
1 beyond the 10-leaf stage. In lines 2, 3, and 4 there was a 2-fold
difference in starch content between young (lowest starch) and old
(highest starch) leaves. Line 1 followed this pattern up to the 10-leaf
stage and then the starch content of the older leaves of line 1 decreased, whereas that of the younger leaves continued to increase
with plant age. By the 22-leaf stage, old leaves of line 1 had less
starch than the young leaves.
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| Figure 9.
Amounts of starch (mmol hexose equivalents
m2) for lines 1 (A), 2 (B), 3 (C), and 4 (D) grown at
high N (, , ) and low N (, , ). Points are means of
four replicates taken during the middle of the photoperiod from the
most recently fully expanded leaves (, ), two leaves below these
leaves (old leaves; , ), and two leaves above these leaves (young
leaves; , ). Error bars represent the maximum SE of
the different-aged leaves.
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To determine whether differences in starch content could account for
the differences in dry weight observed, starch and dry weight were
measured in the same leaf part in plants harvested at the 22-leaf
stage. Starch accounted for between 30% and 55% of the difference in
leaf dry weight, meaning that differences in dry weight were also
caused by differences in structural dry matter (Table
I).
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Table I.
Contribution of starch to dry weight of leaves in
lines 4 and 1 in plants grown at low N at the 22-leaf stage
The mid-leaf is the most recently fully expanded leaf; young leaf is
two leaves above it, and old leaf is two leaves below the mid-leaf.
Data are means ± SE of four replicates.
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DISCUSSION |
Genetic Decrease in PRK Does Not Disrupt N Balance
Our data demonstrate a genetic alteration of photosynthesis
independent of the effects on N or nitrate (Fig. 8, A and B). This is
an important consideration because disruption of plant N balance may in
itself produce large effects on growth and allocation. Nitrate in
particular is an important signal metabolite, regulating shoot-to-root
ratio and C allocation (Scheible et al., 1997). PRK therefore
represents a good target for studies of this nature and a more ideal
one than Rubisco, for which large changes in whole-plant N balance in
plants expressing Rubisco antisense were reported (Quick et al.,
1991a). Furthermore, PRK activity was affected predictably by antisense
in different transgenic lines in leaves of different ages and under
different N regimes (Fig. 1, A-D) further validating the use of the
plant material to examine direct effects between photosynthesis,
growth, and allocation.
The 94% Decrease in PRK Activity Affects Photosynthesis Most at
Low N
At high N the effect of the genetic decrease of PRK on
photosynthesis, even in line 1 with a 94% decrease in PRK activity, was small (Figs. 4, A-D, and 5). At low N there were larger
differences in rates of CO2 assimilation between
line 1 and the other lines (Figs. 4, A-D, and 5B). The explanation for
this is that low N decreases the expression of PRK in itself by 3- to
4-fold compared with high N. A further 94% decrease in PRK by
antisense decreases the activity of PRK sufficiently to impinge more
significantly on the rate of photosynthesis. This effect was then
exaggerated further in older leaves, in which the activities of PRK and
the rates of photosynthesis were very low (Figs. 1A and 4A). An
increase in the activation state of PRK in line 1 (Fig. 3A) was
insufficient to prevent the decrease in photosynthesis. An increase in
activation state, which is a common response to the genetic decrease of
enzymes of different activating mechanisms, e.g. Rubisco (Quick et al., 1991b), NADP-malate dehydrogenase (Trevanion et al., 1997), and Suc-P
synthase (K.-P. Krause, personal communication), would occur in the
case of PRK because of changes in electron transport and consumption of
reducing power within the Calvin cycle (Habash et al., 1996), which
would alter the redox state of thioredoxin and facilitate the
activation of PRK. The increase in Rubisco activation state in these
plants (Fig. 3B) is discussed elsewhere (Paul et al., 1996).
PRK-Limited Photosynthesis at Low N Alters Growth and
Allocation
Low photosynthesis in line 1 at low N resulted in plants that were
smaller than the other lines (Fig. 6B). PRK-limited photosynthesis at
low N not only resulted in smaller plants, but altered allocation of
dry matter within shoots, as measured by higher SLA and LAR, with no
effect on the shoot-to-root ratio (Fig. 6, D, F, and H). Our data
suggest that it is unlikely that photosynthesis has any direct effects
on the shoot-to-root ratio. The changes observed by Fichtner et al.
(1993) in the shoot-to-root ratio in plants with altered Rubisco
content were probably caused by the general disruption of plant N
balance and, particularly, nitrate, which has been shown to be an
important regulator of the shoot-to-root ratio (Scheible et al., 1997).
The inorganic-element content of leaves increased in PRK-limited plants
at low N (Fig. 7, B-G). Accumulation of ions would be facilitated in
slow-growing plants, in which transpiration is relatively insensitive
to a decrease in PRK activity (hence, reduction in water use
efficiency; data not presented). An accumulation of ions would provide
the opportunity for osmotic expansion of leaves and accords with the
data of Fichtner et al. (1993) in establishing a relationship between
SLA and leaf-element content. At the level of the whole plant,
increased SLA could potentially attenuate the effect of decreased
photosynthesis and is associated with changes in the aerial
environment, e.g. light and CO2, which affect the
rate of photosynthesis (Bjorkman, 1981; Garbutt et al., 1990). However,
leaf area per se was unchanged, the increase in SLA reflecting a
decrease in the dry matter of the leaves. This is confirmed by
measurements of whole-plant photosynthesis in line 1 (Fig. 5B), which
was 30% to 35% lower than in the other lines (the same as the
difference in dry weight), demonstrating that changes in leaf
architecture did not prevent an overall decrease in whole-plant
photosynthesis.
Source/Sink Balance and Accumulation of Starch
Accumulation of starch at low N is a well-established
phenomenon (Rufty et al., 1988; Paul and Stitt, 1993; Paul and
Driscoll, 1997) and is interpreted as being a symptom of sink-limited
growth, i.e. the capacity to produce assimilate outweighs the capacity to use it, with surplus assimilate accumulating as starch. Therefore, following this logic, a decrease in the rate of photosynthesis should
be less important to overall biomass production under sink-limited conditions at low N, at which there is much C. However, the opposite was true with PRK-limited photosynthesis at low N, which had a larger effect on growth than at high N (Fig. 6, A and G). This was
partly because the interaction of low N plus antisense pushed PRK
activity lower than at high N, which more markedly affected photosynthesis, but it also indicates that source and sink are less out
of balance at low N than was assumed in our hypothesis. A decrease in
photosynthesis in line 1 at low N resulted in a decrease in starch
content, particularly in older leaves, which beyond the 10-leaf stage
contained less starch than young leaves, in marked contrast to the
normal situation in the wild type and in the other lines (Fig. 9,
A-D). However, starch was still present in large amounts despite the
plants being clearly source limited by low PRK. This seems anomalous,
but it is only so if one assumes that source and sink are out of
balance at this stage of N deficiency and that high starch is
indicative of the current source/sink balance. A large source/sink
imbalance is probably only true of the early stages of N deficiency at
the onset of sugar-induced repression of photosynthetic
activity (Paul and Driscoll, 1997). High starch may be more a
reflection of this period of imbalance than of the current status, and
starch persists simply because of low sink activity rather than an
ongoing large source/sink imbalance.
It is worth comparing our results with those of similar experiments
carried out on Rubisco transgenic plants (Quick et al., 1991a; Fichtner
et al., 1993). Here there was almost complete allocation of C
away from starch in transgenic plants with decreased Rubisco activity
at low N. Starch content responded more strongly to the changed
source/sink balance because of Rubisco antisense. Allocation of C away
from starch probably accounted for the observation that structural dry
weight did not differ between the wild-type and the Rubisco transgenic
plants. In line 1 of the PRK transgenic plants structural dry weight
was lower than in the other lines. A possible explanation for the
difference in C allocation may be that a decrease in Rubisco content,
at least in the early phase of N defi-ciency, releases a large amount
of N for use elsewhere in the plant (Paul and Stitt, 1993). A decrease
in the amount of PRK, however, will not result in a large
redistribution of N because PRK accounts for only a fraction of the N
within a leaf (Evans, 1989). Thus, decreased photosynthesis in
PRK antisense plants occurs without any N redistribution. Strong
interactions between starch metabolism and nitrate and N
metabolism have been demonstrated (Scheible et al., 1997). N
balance was clearly perturbed in Rubisco transgenic plants, which in
itself may have affected starch metabolism. It is also possible that
internal N signals generated through mobilization of Rubisco
influence the way long-term C stores are managed, and the extension of
this in Rubisco transgenic plants may have ensured the more
effective partitioning of C away from starch in these plants. In
conclusion, genetic decrease of PRK goes beyond the normal
acclimation of plants to low N, and the resulting decrease in
photosynthesis limits biomass production under conditions normally
regarded as sink limited.
| |
FOOTNOTES |
1
IACR receives grant-aided support from the
Biotechnology and Biological Sciences Research Council (BBSRC) of the
United Kingdom. This work was supported by a grant from the BBSRC Clean
Technology Initiative.
*
Corresponding author; e-mail matthew.paul{at}bbsrc.ac.uk; fax
44-1582-760981.
Received September 18, 1998;
accepted December 12, 1998.
| |
ABBREVIATIONS |
Abbreviations:
LAR, leaf area ratio.
PRK, phosphoribulokinase.
SLA, specific leaf area.
| |
ACKNOWLEDGMENTS |
We thank Steven Dunn for help and advice on the determination of
nitrate; Maureen Birdsey, Jeanne Day, and Adrian Crosland for
performing the analysis of elements; and Roger Leigh and Steve Trevanion for their comments on a draft of the manuscript.
| |
LITERATURE CITED |
Bjorkman O
(1981)
Responses to different quantum flux densities.
In
OL Lange,
PS Nobel,
CB Osmond,
H Ziegler,
eds, Encyclopedia of Plant Physiology, Vol 12A: Physiological Plant Ecology. I. Responses to the Physical Environment.
Springer-Verlag, Berlin, pp 57-107
Chapin FS,
Schulze ED,
Monney HA
(1990)
The ecology and economics of storage in plants.
Annu Rev Ecol Syst
21:
423-447
[CrossRef][ISI]
Evans JR
(1989)
Photosynthesis and nitrogen relationships in leaves of C3 plants.
Oecologia
78:
9-19
[CrossRef][ISI]
Fichtner K,
Quick WP,
Schulze E-D,
Mooney HA,
Rodermel SR,
Bogorad L,
Stitt M
(1993)
Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with antisense rbcS. V. Relationship between photosynthetic rate, storage strategy, biomass allocation and vegetative plant growth at three different nitrogen supplies.
Planta
190:
1-9
Garbutt K,
Williams WE,
Bazzaz FA
(1990)
Analysis of the differential response of five annuals to elevated CO2 during growth.
Ecology
71:
1185-1194
Habash DZ,
Parry MAJ,
Parmar S,
Paul MJ,
Driscoll S,
Knight J,
Gray JC,
Lawlor DW
(1996)
The regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity.
Photosynth Res
49:
159-167
[CrossRef]
Jones MGK,
Outlaw WH,
Lowry OH
(1977)
Enzymic assay of 107 to 104 moles of sucrose in plant tissues.
Plant Physiol
60:
379-383
[Abstract/Free Full Text]
Kagawa T (1982) Isolation and purification of ribulose-5-phosphate
kinase from Nicotiana glutinosa. In M Edelman, KB
Hallick, N-H Chua, eds, Methods in Chloroplast Molecular Biology.
Elsevier Biomedical Press, Amsterdam, pp 695-705
Knight JS, Gray JC (1994) Transgenic Plants as a Tool for the
Study of Photosynthetic Carbon Assimilation: Trends in Photosynthesis
Research. Intercept, Andover, UK, pp 267-277
Leegood RC
(1990)
Enzymes of the Calvin cycle.
In
PJ Lea,
eds, Methods in Plant Biochemistry, Vol 3.
Academic Press, London, pp 16-20
Misko TP,
Schilling RJ,
Salvemini D,
Moore WM,
Currie MG
(1993)
A fluorometric assay for the measurement of nitrate in biological samples.
Anal Biochem
214:
11-16
[CrossRef][ISI][Medline]
Paul MJ,
Andralojc PJ,
Banks FM,
Parry MAJ,
Knight JS,
Gray JC,
Keys AJ
(1996)
Altered Rubisco activity and amounts of a daytime tight-binding inhibitor in transgenic tobacco expressing limiting amounts of phosphoribulokinase.
J Exp Bot
47:
1963-1966
Paul MJ,
Driscoll SP
(1997)
Sugar repression of photosynthesis: the role of carbohydrates in signalling nitrogen deficiency through source/sink imbalance.
Plant Cell Environ
20:
110-116
[CrossRef]
Paul MJ,
Knight JS,
Habash D,
Parry MAJ,
Lawlor DW,
Barnes SA,
Loynes A,
Gray JC
(1995)
Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth in low irradiance.
Plant J
7:
535-542
[CrossRef][ISI]
Paul MJ,
Stitt M
(1993)
Effects of nitrogen and phosphorus deficiencies on levels of carbohydrates, respiratory enzymes and metabolites in seedlings of tobacco and their response to exogenous sucrose.
Plant Cell Environ
16:
1047-1057
[CrossRef]
Poorter H,
Remkes C
(1990)
Leaf area ratio and net assimilation rate of 24 wild species differing in relative growth rate.
Oecologia
83:
855-859
Poorter H,
Remkes C,
Lambers H
(1990)
Carbon and nitrogen economy of 24 wild species differing in relative growth rate.
Plant Physiol
73:
553-559
Quick WP,
Schurr U,
Fichtner K,
Schulze E-D,
Rodermel SR,
Bogorad L,
Stitt M
(1991a)
The impact of decreased Rubisco on photosynthesis, growth and storage in tobacco plants which have been transformed with antisense rbcS.
Plant J
1:
51-58
Quick WP,
Schurr U,
Scheibe R,
Schulze E-D,
Rodermel SR,
Bogorad L,
Stitt M
(1991b)
Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with antisense rbcS. I. Impact on photosynthesis in ambient growth conditions.
Planta
183:
542-554
[ISI]
Rufty TW,
Huber SC,
Volk RJ
(1988)
Alterations in leaf carbohydrate metabolism in response to nitrogen stress.
Plant Physiol
88:
725-730
[Abstract/Free Full Text]
Scheible W-R,
Lauerer M,
Schulze E-D,
Caboche M,
Stitt M
(1997)
Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco.
Plant J
11:
671-691
[CrossRef]
Sonnewald U,
Brauer M,
von Schawaen A,
Stitt M,
Willmitzer L
(1991)
Transgenic tobacco plants expressing yeast-derived invertase in either cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions.
Plant J
1:
95-100
[CrossRef][ISI][Medline]
Stitt M,
Schulze D
(1994)
Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology.
Plant Cell Environ
17:
465-487
[CrossRef]
Trevanion SJ,
Furbank RT,
Ashton AR
(1997)
NADP-malate dehydrogenase in the C4 plant Flaveria bidentis.
Plant Physiol
113:
1153-1165
[Abstract]
Verdon CP,
Burton BA,
Prior RL
(1995)
Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP+ when the Griess reaction is used to assay for nitrate.
Anal Biochem
224:
502-508
[CrossRef][ISI][Medline]
von Caemmerer S,
Farquhar GD
(1981)
Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves.
Planta
153:
376-387
[CrossRef][ISI]
Woodrow IE,
Berry JA
(1988)
Enzymatic regulation of photosynthetic carbon dioxide fixation in C3 plants.
Annu Rev Plant Physiol Plant Mol Biol
39:
533-594
[ISI]
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Abstract
Introduction