Transgenic Plants and Plant
Biochemistry
Society/Host Colloquium Organized and Edited by P. J. Lea (University of Lancaster). 65 I s t Meeting held at
University of Lancaster, I 3- I 4 July I994
Analysis of transgenic tobacco plants containing varying amounts of ribulose- I ,5bisphosphate carboxylase/oxygenase
W. Paul Quick
Department of Animal and Plant Sciences, University of Sheffield, P.O. Box 601, Sheffield SIO 2UQ, U.K.
The fixation of CO, during photosynthesis is catalysed by ribulose-1,s-bisphosphate carboxylase/
oxygenase (Rubisco). This enzyme is widely thought
to exert considerable control on the rate of photosynthesis especially in C, plants [ 1,2]. Rubisco has
a low specific activity and a poor affinity for CO,,
which in current atmospheric conditions requires
that Rubisco operates below or close to its K,,, for
CO,. Rubisco also catalyses a competitive oxygenase reaction which can account for up to 30% of its
activity and leads to the production of Z-phosphoglycollate. This product is then further metabolized
back to 3-phosphoglycerate via the photorespiratory pathway, resulting in a loss of CO, and Nti,
and the consumption of ATP and reducing equivalents.
Two other considerations arise as a result of
the relative inefficiency of Rubisco. Firstly, large
quantities of Rubisco protein are produced in
photosynthetic tissues, which can account for
30-50% of leaf-soluble protein. The catalytic-site
concentration in the chloroplast stroma is typically
within the range 3-10mM, close to that found in
pure Kubisco crystals and more typical of values
normally associated with low-molecular-mass
metabolites. This represents a large investment of
nitrogen (a scarce nutrient) in a single protein that
might otherwise be used to sustain other aspects of
plant growth. Secondly, the low concentration of
atmospheric CO, requires that leaf resistance to
C02diffusion be low if internal CO, concentrations
are to be maintained during periods of active photosynthesis. This is achieved by increasing stomatal
aperture and hence stomatal conductance. However, this also results in the evaporative loss of
water from the plant, often at rates several hundredfold greater than that of photosynthesis. Water and
nitrogen availability are of major importance to
plants, limiting growth in many environments, and
often outweigh purely photosynthetic considerations [ 3 - 5 ] .
The activity of this enzyme is also highly
regulated. both at the level of gene expression and
through biochemical regulation. Activation of
Rubisco is brought about by the binding of CO,
and M g ’ to a lysine residue close to the active site
(carbamylation). This process is mediated in vivo
by an enzyme, Kubisco activase, that requires ATP
and which is inhibited by ADP. The proportion of
Rubisco in the active form has been shown to follow closely the steady-state rate of photosynthesis,
and this is probably mediated via changes in the
ATPIADP ratio of the chloroplast stroma [6].
Rubisco activity is also inhibited by the tight binding of a naturally occurring inhibitor, carboxyarabinitol- 1-phosphate, to the active site. This
compound is found in high concentrations in some
plants during the night and its concentration
declines upon illumination. The precise details of
how this compound is removed from the active site,
the regulation of its synthesis and degradation and
how this is co-ordinated with Rubisco activase are
still unclear. Both of these proceses do, however,
serve to match the activity of Rubisco with the prevailing rate of photosynthesis [ 6 ] .
The amount of Rubisco found in a leaf varies
markedly according to the prevailing growth conditions and availability of nutrients. For example,
there is generally a good correlation between photosynthetic capacity and leaf nitrogen content, largely
mediated through changes in the amount of photosynthetic enzymes, including Kubisco [7]. The light
intensity during growth has a strong regulatory
Abbreviations used: IW),photon flux density; I’SII.
photosystem II; @l’Sll. quantum efficiency of I’SII.
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Biochemical Society Transactions
900
effect on the amount of Rubisco protein found in a
leaf; an increase in growth photon flux density
(PFD) generally results in increased amounts of
Rubisco protein [8]. Changes in the Rubisco content of mature leaves have been shown to occur
over relatively short time periods [ 1-2 days),
involving changes in protein and mRNA levels [9].
The manipulation of source or sink activity can also
mediate changes in the photosynthetic composition
of leaves [ 101. Generally, manipulations that result
in a decreased requirement for photosynthate cause
a reduction in the photosynthetic capacity of leaves
and visa versa. More recently, carbohydrates have
been shown to have a direct effect on the amount of
several photosynthetic proteins [ l l ] and to act at
the level of gene expression [ 121. The activity and
amount of Rubisco is tightly controlled by a variety
of environmental and developmental signals both in
the short and long term. The precise control exerted
by Rubisco is therefore likely to be complex and
variable depending on many environmental factors.
Transgenic tobacco plants that contain antisense DNA for the small subunit of Rubisco have
reduced amounts of Rubisco protein [ 131. We have
used these plants to evaluate directly the consequences of altered amounts of Rubisco protein on
plant photosynthesis and growth [ 14-20]. Further,
the availability of a variety of plants that exhibit a
range of Rubisco activities has allowed us to quantify the control Rubisco exerts on photosynthesis
(CL) using metabolic control analysis [21]. A
simple method to establish the control exerted by a
particular enzyme on the flux through a pathway is
to measure how the flux is altered by small changes
in the amount of enzyme. This has been made possible experimentally by the rapid development of
plant molecular biology and the availability of a
range of transgenic plants with altered amounts of
specific enzymes.
This approach, first formalized by Kascer and
Burns [ 221 and Heinrich and Rapaport [231, allows
the calculation of a flux control coeficient (Ck):
summarizes some of our recent findings with these
transgenic tobacco plants, many of which are
already published [ 14-20].
The ability of plants to acclimatize photosynthetically to a variety of environmental conditions
through changes either in morphology and/or protein complement requires that photosynthetic
measurements be determined in conditions that are
relevant to the environmental growth conditions.
Plants used in our experiments were grown in
growth cabinets where the environment is carefully
controlled; this allowed us to vary the growth environment, to examine the control exerted by
Rubisco in the ambient environment and to perturb
the environment to assess how control is affected by
short-term environmental changes. Data presented
in Figure 1 were obtained from plants grown at a
relatively low, photosynthetically active PFD of
Photosynthesis measured from wild-type and transgenic plants as a function of the Rubisco activity
measured in the first fully expanded leaf
Photosynthetic CO, fixation was determined in the ambient
growth environment, 20°C and 300pmol.s-'.m-'. PFD ( 0 ) or
at altered PFD; 100 (A)or 1000pmol.s-'*m-2 (0). The rate
of photosynthetic oxygen evolution measured from leaf discs
in an oxygen electrode at 5kPa CO,. 20°C and
l 0 0 0 p m o l . ~ - ' . m - ~PFD
. ( V )is also shown as a measure of
photosyntheticcapacity.
35
I
I
I
1
30
40
v
30
h
-E
25
I
-?0
5.m
20
3
5
f
U
I5
c0:
a
L
0
where dE/E is the fractional change in the amount
of enzyme ( E ) and dJ/J is the resultant fractional
change in flux (J)through the pathway. For linear
metabolic pathways, CL: can range from 0 to 1. A
value approaching zero indicates that an enzyme
has little control on the flux through the pathway
whereas a value approaching 1 indicates that the
enzyme has a major control on flux. This paper
Volume 22
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2
10
5
,_By
0
10
20
Rubisco activity (pmo1.s
50
*)
Transgenic Plants and Plant Biochemistry
300 pmol*s-’*m-’. Each data point represents a
single measurement made on an individual plant.
Photosynthesis was then measured in conditions
that approximate to the growth environment
(Figure I . 0 ) . The data show that, as Rubisco was
progressively reduced from wild-type activities, the
rate of photosynthesis declined in a biphasic
manner. There was an initial shallow dependence of
photosynthesis on Rubisco activity followed by a
very steep dependence when Rubisco was reduced
by >40% of the average wild-type value.
Measurement of the slope of this curve at the average wild-type value gives an estimate of the flux
control coeficient of Rubisco for photosynthesis in
wild-type plants. A useful method to obtain this
value is either to fit a linear regression to data with
values of F: close to the wild-type or to obtain the
‘best tit’ of a rectangular hyperbolic function, a E /
( b + E). The control coeficient can then be determined for any value of E by differentiating the
previous equation, to give ab/(E+ b)’, and multiplying by a scaling factor (W’j’)[24]. In the ambient
growth conditions, the latter technique provides a
value of 0.25 and indicates that Rubisco exerts only
a partial control on the rate of photosynthesis in the
growth environment. Plants with < 40% less
Rubisco have similar rates of photosynthesis to
wild-type plants. This is a result of biochemical
regulation. Wild-type plants maintain Rubisco at
50% of full activation in these conditions whereas
plants with reduced Rubisco maintain a higher activation state (approaching 100%);this indicates that
wild-type plants contain considerably more
Kubisco than is required to sustain photosynthesis
in the ambient growth environment [ 141. Metabolite
measurements showed that increased activation of
Rubisco in transgenic plants was associated with an
increased ATI)/AI)P ratio, and analysis of chlorophyll fluorescence quenching showed a large
increase in non-photochemical quenching, indicative of a large transthylakoid proton gradient [ 141.
These results are in agreement with current models
of Rubisco regulation by Rubisco activase [ h ] .
For this type of analysis to be valid, it is essential to show that only the amount of Rubisco has
been altered as a result of the genetic manipulation.
Rubisco is quantified in terms of V,ll,x activity in
this paper for clarity. €lowever, we have carefully
shown that activity is directly related to the amount
of protein, as required for flux control analysis [ 141.
Measurement of several chloroplastic and cytosolic
enzymes revealed that significant decreases in activity only occur when the amount of Rubisco is
reduced by > 50% 11.11. Interestingly. it is at this
-
point that plants begin to show phenotypic changes
(plants are smaller and have reduced chlorophyll).
Only data where Rubisco is altered by < 50% were
used for control analysis. Further confirmation of
the lack of pleiotropic effects was obtained from
measurements of photosynthetic capacity (Figure 1,
V). CO, was provided at saturating partial pressures (5 kPa) to minimize the control exerted by
Rubisco and the maximum rate of photosynthesis
was measured in an oxygen electrode at saturating
light (1000 pmol*s-’*m-’). The data show that
transgenic plants with considerable reductions in
Rubisco activity ( < 50%) were able to sustain much
higher rates of photosynthesis in these conditions
than those measured in the ambient conditions
(Figure 1, 0 ) or in ambient C 0 2 and saturating light
(Figure 1, 0). This is direct evidence that other
aspects of photosynthetic metabolism were not
significantly influenced by the genetic manipulation.
In the field, plants experience a wide variety of
growth environments that can fluctuate over both
short (minutes-hours) and longer (days-weeks)
timescales. The ability to respond to these changes
is essential for efficient photosynthesis. These transgenic plants can also be used to reveal how shortterm environmental changes affect the control
exerted by Rubisco on the rate of photosynthesis.
This is illustrated in Figure 1, where the light
intensity was either reduced below ambient (Figure
I , A) or increased to saturating intensities (Figure 1,
0). The data show that the control exerted by
Rubisco is altered markedly by light intensity from
a very low value in low-intensity light (C[= 0.003)
to a very high value in high-intensity light
(CK= 0.76). The apparent ‘excess’ Rubisco found in
wild-type plants thus confers a major advantage as
it allows the rate of photosynthesis to increase when
the light intensity is increased. Plants with reduced
Rubisco activity are largely light saturated in the
ambient growth conditions and do not have the
metabolic flexibility of wild-type plants.
Many plants acclimatize to growth in altered
light environments by changing the amounts of a
range of photosynthetic enzymes as well as by
changes in leaf and whole-plant morphology. Figure
2 shows data obtained from tobacco grown in a
range of light environments. The mean Rubisco
content found in mature leaves of wild-type plants
increases with growth PFL) (Figure 2a). Figure 2(b)
shows the estimated CK for Rubisco on photosynthesis in plants grown either at 300 p mo l* s -’-m -L
PFD and subjected to short-term changes in PFL)
(Figure 2, m) or grown at different PFIIs and determined in the ambient conditions (Figure 2, *).
I994
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Biochemical Society Transactions
902
Mean activity of Rubisco measured from wild-type
plants as a function of their growth light intensity ( a )
and variation in CL with changes in PFD ( b ) for
plants either grown at different PFD and measured
in the growth environment ( 0 ) or grown at
300pmol*s-'*m-2PFD and measured at a variety of
PFDs (B)
*
-
120
1
1.0
r
0.8
t
I
1
1
1
1
I
Non-acclimatized
0.6
/'
P(r
u
r
-
0.4
0.2
t
0.0
0
200
400
600
800
1000
1200
PFD (pmo1.s ' . m *)
Short-term increases in PFD led to a progressive
increase in Cl; from almost zero in low-intensity to
values approaching 0.8 at lOOOpmol.s-'*m-'.
Plants grown at high PFI) produce more Rubisco
and hence the control exerted by Rubisco on photosynthesis is reduced (from 0.8 to 0.25 at
1000 pmol 's - -m- ') [ 19J. Acclimatization resulted
in reduced control for Rubisco in high PFI) and
ensured that control is not located on a single
enzyme but rather is shared among the various
components of photosynthesis. Similar results were
obtained with plants grown in environments of elevated CO,; short-term increases in CO, concentration reduce the control exerted by Rubisco on
photosynthesis but in the longer term the amount of
Rubisco is reduced and control is increased (R.
Alred and W. P. Quick, unpublished work). Decreased availability of nitrogen during growth, however, gave the opposite result to growth in altered
regimes of light or CO,. As the amount of nitrogen
supplied was reduced, the amount of Rubisco in the
leaves was also reduced but the control exerted by
Rubisco on photosynthesis was increased (Cl;
increased from 0.2 to 0.58 when [N] was reduced
from 5.0 to 0.1 mM) [ 17,181. It appears that one
strategy to conserve nitrogen in conditions of
limited supply is to reduce specifically the amount
of Rubisco; given the large quantities of this
enzyme, this would seem a suitable strategy for
ensuring efficient use of nitrogen.
Rubisco also catalyses an oxygenation reaction which leads to a loss of CO, and the consumption of ATP and reducing equivalents during the
process of photorespiration. Although this reaction
is often regarded as wasteful, there is some evidence
in the literature that photorespiration may be an
important mechanism for dissipation of light energy
in conditions that lead to reduced internal CO' concentrations (eg. water stress and high temperatures)
[25]. The rate of electron transport through photosystem I1 (PSII) can be estimated from analysis of
chlorophyll fluorescence and calculation of the
quantum efficiency of PSI1 (@PSII)1261. Previous
studies have shown a good correlation between
@PSI1and the rate of CO, fixation in both photorespiratory and non-photorespiratory conditions
[27,28]. The results obtained from the simultaneous
measurement of photosynthesis and chlorophyll
fluorescence measured at moderate PFI)
(2 1 5 p m *s - *m- ') at various partial pressures of
CO, and in a range of plants with differing Rubisco
contents are summarized in Figure 3. The estimated
'
4y-2
.
:
>*
.':
Variation in CL (A) and C? ( 0 ) as a function of the
calculated internal CO, concentration of the leaf
Photosynthesis and chlorophyll fluorescence were measured
simultaneously at 215pmol~s-'.m-' PFD and 20°C.
'
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0.9
0.8
U
c
0.7
f
0
0.6
.-U
-8
e
U
0.5
E
0.4
-
0.3
8
X
Y
0.2
0. I
0.0
'
0
20
40
60
80
Internal CO, partial pressure (Pa)
I
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Transgenic Plants and Plant Biochemistry
flux control coefficient of Kubisco for photosynthesis (Ck) and for electron transport through PSI1
(C:) were calculated as described previously.
Values for Cl; at low partial pressures of C02 are
not included due to inaccuracies that occur when
measuring very low rates of photosynthesis. The
data show that as the partial pressure of CO, is
lowered, the control exerted by Rubisco on photosynthesis is increased, as would be predicted for
this enzyme. T h e control that Rubisco exerts on
electron transport shows a parallel increase which
attains very high values at CO, partial pressures
below 10Pa. This suggests that Rubisco has a
major role in maintaining electron transport at low
CO, and indicates that photorespiration is the major
pathway through which this occurs, rather than, for
example, the Mehler reaction. Increased amounts of
Rubisco are of clear advantage in conditions that
lead to low mesophyll C 0 2concentration.
In conclusion, transgenic plants provide an
excellent experimental system for the analysis of
metabolic pathways. The availability of plants with a
wide range in the amount of a particular enzyme are
particularly suited for quantitative metabolic control
analysis. Our results show that the control exerted
by Kubisco on photosynthesis is often low when
measured in ambient conditions. and that this control is strongly influenced by alterations to these
conditions in both the short and the long term. This
emphasizes the need for care when choosing conditions for analysis, which must be related in some
way to the plant’s growth environment. These
results also show that the oxygenase activity of
Rubisco may have a major role in maintaining
electron transport during periods of water stress, a
feature even of irrigated crops on hot sunny days.
The results obtained from this particular set of
tobacco plants with altered Rubisco activity have
given many insights not only into the role of
Rubisco in photosynthetic metabolism but also, in
other work [ 16-20], into the integration of photosynthesis with the growth and development of the
plant.
I thank I)r. K. C. I,eegood and Ilr. G. A. F. tlendry for
their critical reading of this paper.
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Received 22 July 1994
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