Expressing an RbcS Antisense Gene in - Plant Physiology

Plant Physiol. (1 997) 11 5: 11 63-1 174

Expressing an RbcS Antisense Gene in Transgenic flaveria bidentis Leads to an lncreased Quantum Requirement for

CO, Fixed in Photosystems I and II

Katharina Siebke*, Susanne von Caemmerer, Murray Badger, and Robert T. Furbank

lnstitut für Botanik, Schlossgarten 3, D-48149 Münster, Germany (K.S.); Research School of Biological Sciences, Molecular Plant Physiology Group, G.P.O. Box 475, Canberra, ACT 2601, Australia (S.V.C., M.B.); and

Commonwealth Scientific and Industrial Research Organization Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia (R.T.F.)

It was previously shown with concurrent measurements of gas exchange and carbon isotope discrimination that the reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase by an antisense gene construct in transgenic Haveria bidentis (a C, species) leads to reduced CO, assimilation rates, increased bundle-sheath CO, concentration, and leakiness (defined as the ratio of CO, leakage to the rate of C, acid decarboxylation; S. von Caemmerer, A. Millegate, C.D. Farquhar, R.T. Furbank [1997] Plant Physiol 113: 469-477). lncreased leakiness in the transformants should result in an increased ATP requirement per mole of CO, fixed and a change in the ATP-to-NADPH demand. To investigate this, we compared measurements of the quantum yield of photosystem I and II (QpSl and QPsII) with the quantum yield of CO, fixation (aco2) in control and transgenic F. bidentis plants in various conditions. Both @ps,/@coz

and @psII/@co, increased with a decrease in ribulose-1,S- bisphosphate carboxylase/oxygenase content, confirming an increase in leakiness. I n the wild type the ratio of QPsI to QPsII was constant at different irradiances but increased with irradiance in the transformants, suggesting that cyclic electron transport may be higher in the transformants. To evaluate the relative contribution of cyclic or linear electron transport to extra ATP generation, we developed a model that links leakiness, ATP/NADP requirements, and quantum yields. Despite some uncertainties in the light distribution between photosystem I and II, we conclude from the increase of @psI,/@co2 in the transformants that cyclic electron transport is not solely responsible for ATP generation without NADPH production.

The C, plant Flaveria bidentis was previously transformed with an antisense gene construct inhibiting the expression of Rubisco, whereas the expression of other photosynthetic enzymes was largely unaffected (Furbank et al., 1996). These transformants are a useful tool for examining the role of Rubisco in determining regulatory processes in C, photosynthesis in vivo. Concurrent measurements of gas exchange and C isotope discrimination have shown that the reduction of Rubisco led to reduced CO, assimilation rates, increased bundle-sheath CO, concentration, and leakiness, defined as the ratio of CO, leakage out of the bundle sheath to the rate of C, acid decarboxylation (von

* Corresponding author; e-mail siebkeauni-muenster.de; fax 49 -251-832-3823.

Caemmerer et al., 1997). In this paper we examine how this increased leakiness affects the energy requirement for photosynthesis.

F . bidentis is an NADP-ME-type C, plant (Moore et al., 1989) that transfers mainly malate (60-65%) but also aspartate (3540%) between mesophyll and bundle-sheath cells (Meister et al., 1996). Neglecting RuBP oxygenation, one would expect that F. bidentis minimally requires 5 mo1 of ATP and 2 mo1 of NADPH per mo1 of CO, fixed (Hatch, 1987). Two ATP equivalents are used by pyruvate Pi diki- nase, producing PEP, which is carboxylated to oxaloacetic acid in the mesophyll; 1 mo1 of ATP is used by phospho- ribulokinase, producing RuBP; 2 mo1 of ATP and 2 mo1 of NADPH are used by phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase for the production of 2 mo1 of dihydroxyacetone phosphates from 2 mo1 of PGA.

CO, leakage out of the bundle sheath increases the amount of ATP used in the C, cycle by pyruvate Pi diki- nase per mole of CO, fixed (Hatch, 1987). The NADPH used for the formation of malate from oxaloacetic acid either in the mesophyll chloroplasts or in the bundle- sheath chloroplasts (if aspartate has been transferred) is regenerated during the decarboxylation in the bundle- sheath chloroplasts by NADP-ME. Since PGA and dihydroxyacetone phosphate can be transferred between bundle-sheath and mesophyll cells in C, plants, the relative consumption of NADPH per ATP in the mesophyll or

Abbreviations: A, P700+ A,,, during steady-state photosynthesis; A,,,, P700+ absorbance level with maximal oxidation of P700; A , P700+ absorbance level with maximal reduction of P700; A,,,, P700+ absorbance in a saturating light pulse during steady-state photosynthesis; F , fluorescence yield of a dark-adapted leaf; Fo', fluorescence of a light-adapted leaf measured in the presence of far-red light only; F,, maximal fluorescence of a dark-adapted leaf; FM', maximal fluorescence of a light-adapted leaf; FpSr, fraction of fluorescence signal attributed to PSI; F,, fluorescence during steady-state photosynthesis; Qc0,, CO, fixed per absorbed quantum; @,,SI, quantum yield of PSI; @'psII, quantum yield of PSII; ME, malic enzyme I/I, leakiness (ratio of CO, leakage to rate of C, acid decarboxylation); P700, reaction center chlorophyll of PSI; PGA, 3-phosphoglycerate; QA, primary electron acceptor; qN, non- photochemical quenching; qp, photochemical quenching; R,, mito- chondrial respiration; RuBP, ribulose-1,5-bisphosphate.

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1164 Siebke et al. Plant Physiol. Vol. 11 5, 1997

bundle-sheath chloroplasts can be flexible. For Zea mays, an NADP-ME plant with little PSII activity in the bundle sheath, this transfer of PGA to the mesophyll cells is crucial (Furbank and Badger, 1983). In contrast, bundle-sheath cells of F. bidentis can sustain high rates of whole-chain electron transport and the proportion of PGA reduced in the mesophyll may not be as high as in maize (Meister et al., 1996).

In the transgenic F. bidentis plants with reduced Rubisco content, the rate of CO, fixation in the bundle sheath is inhibited, and both ATP and NADPH requirements are decreased. However, since the C, cycle activity is not ap- preciably down-regulated to match the decreased C, cycle activity, the leakiness and therefore the requirement of ATE’ per mole of CO, fixed increases (von Caemmerer et al., 1997). Consequently, the flux through PSII could be down-regulated to match the leve1 of NADPH consumption, and the change in thylakoid pH necessary for the production of additional ATP could be supplied via cyclic electron transport around PSI without NADPH production (Asada et al., 1993). Alternatively, there is strong evidence that in C, plants additional ATP can be produced without NADPH production by an increased linear electron flow to O, (Furbank et al., 1983). In this paper we examine how the change in ATP and NADPH demand per CO, fixed in transgenic F. bidentis with reduced amounts of Rubisco affects the quantum use of PSI and PSII.

MATERIALS AND METHODS

Plant Culture

Haveria bidentis was previously transformed with an antisense RNA construct targeted to the nuclear-encoded gene for the small subunit of Rubisco fFurbank et al., 1996). Plants used were from the T, progeny of primary transformant 136-13 (four inserts) and control transformants (transformed with a GUS construct) and were the same lines used by von Caemmerer et al. (1997) to measure C isotope discrimination. Plants were grown as described by von Caemmerer et al. (1997) in a naturally lit greenhouse in February, 1996. They were given a complete nutrient solu- tion containing 12 mM NO,- (Hewitt and Smith, 1975) three times a week.

Cas-Exchange Measurements

Gas exchange was measured with a portable photosynthesis system (model 6400, Li-Cor, Lincoln, NE). Air temperature was 20 or 25”C, and humidity varied between 50 and 80%. X, of the leaf was measured after 30 min of dark adaptation. A special cuvette was used to fit a polyfurcated fiber optic connecting the different light sources and measuring beams to ensure equal distribution of illumination and detection of light over the leaf area. The actinic light source (a tungsten halogen lamp) was equipped with a Calflex X-filter to cut off light above 800 nm (Balzers, Liechtenstein). The QpsI and @ps,, were measured sequen- tially during steady-state photosynthesis, with a time delay of about 3 min.

Chlorophyll a Fluorescence

QpsII was determined by chlorophyll a fluorescence, a noninvasive optical tool with high diagnostic value in photosynthesis research (Krause and Weis, 1991). The different fluorescence quenching parameters can be determined using pulse-modulated fluorometry with a brief, saturating light pulse (PAM 101, Walz, Effeltrich, Germany; Schreiber et al., 1986). We defined the different fluorescence levels as suggested by van Kooten and Snel (1990). The qN of chlorophyll u fluorescence is determined by the equation: qN = 1 - (F,’ - Fo’)/ (FM - Fo); the qp is given by: qp = (F,’ - Fs)/(FM’ - Fo’). QpsII = (FM‘ - Fs)/FM’ (Genty et al., 1989). In this expression the contribution of PSI fluorescence to the measuring signal is neglected. It depends on the spectral region of the detected light.

Especially when plants with a high proportion of PSI relative to PSII, such as C, plants, are measured, it is important to take PSI fluorescence into account (Genty et al., 1990; Pfündel, 1995). PSI fluorescence, unlike PSII fluorescence, is constant and is not significantly quenched (Genty et al., 1990). In a fully dark-adapted F. bidentis leaf, the QPsII was assumed throughout to be 0.877 (Pfündel, 1995). The contribution of FFsI is given by F , - ( F , - F o ) / 0.877. The value obtained for PSI fluorescence was sub- tracted from a11 fluorescence levels.

This correction procedure for the emitter detector unit (model 101-ED, red LED 650 nm, detection region 700-800 nm, Walz) was tested with fluorescence measurements using a blue emitter LED (model US-L450, Walz) equipped with a blue filter to eliminate the red region (B-51, Balzers), together with a detector unit (model ED-lOlUS/D, Walz) protected by a red filter (RG-665 filter, Schott, Mainz, Ger- many) and a short-wave-pass filter (LS-700, Corion, Hol- liston, MA), resulting in a detection region of 650 to 700 nm, where no contribution of PSI fluorescence occurs. The signal with the blue light-emitting dioxde had a poor signal-to-noise ratio compared with the signal with the usual emitter detector unit. We therefore evaluated the average signal of four to five saturating flashes taken in the steady state with about 3-min intervals between the flashes. The difference between the corrected and uncor- rected values increases from 9% for high to 35% for low QPsII values.

P700+ Absorbance

The QpsI can be determined via P700+ absorbance changes at A,,, (Schreiber et al., 1988; Klughammer and Schreiber, 1994). For the oxidation of PSI, leaves were first illuminated with a far-red light from a tungsten halogen lamp filtered through an RG 9 (Schott) and a Calflex X (Balzers) filter together with a photodiode (102-FR, Walz; total far-red light: 120 pmol m-’ s-’) and then fully oxidized with a saturating white-light pulse (1 s, 9500 pmol m--2 s-l ). The saturating tungsten halogen light-pulse lamp was equipped with an electromagnetic shutter (UniB- litz, Vincent Associates, New York) and short-pass filters (DT-Cyan and Calflex X, Balzers) for fast on/off kinetics.

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Quantum Requirement of CO, Fixation in Transgenic %veria bidentis

QFsI was calculated as (A,,, - A)/(AmaX - Ao). For the measurement of A,,,, we used a slightly different procedure than that used by Klughammer and Schreiber (1994; see "Appendix A ) .

Light lntensity

Light intensities were measured with a quantum sensor (Li-Cor) or in the case of the far-red light with a pyranom- eter (Li-Cor), which was calibrated with the quantum sensor at different wavelengths using the sensitivity curves of the sensors.

Biochemical Measurements

Chlorophyll concentration was determined in acetone according to the method of Porra et al. (1989). Leaf absorptance was calculated by the formula: absorptance = Chl/(Chl + 0.076 mmol m-') (Evans, 1993). This formula was confirmed for F. bidentis with an Ulbricht integrating sphere using a tungsten halogen lamp and a quantum sensor (Li-Cor).

After gas-exchange measurements, leaf discs (1.61 cm') were collected, frozen in liquid N,, and stored at -80°C for subsequent analysis. Rubisco content was determined from a [14C]carboxyarbabinitol bisphosphate-binding assay (Butz and Sharkey, 1989; Mate et al., 1993), and soluble protein was quantified using Coomassie Plus reagent (Pierce) on the same extracts.

RESULTS AND DISCUSSION

Relationship between Photosynthesis and Rubisco Content of Leaves

Dependence of CO, Assimilation Rate on Rubisco Content

Figure 1A shows the dependence of the CO, assimilation rate (measured at a light intensity of 620 pmol quanta m-' s-l) on the Rubisco content. Measurements were made on different plants from the T, progeny of primary transformants 136-13 and a control transformant. To calculate the gross CO, fixation rate, the R, was added to the net CO, assimilation rate, and RuBP oxygenation was neglected. It was assumed that the R, in the light equals the R, in the dark, although it is likely to be lower during illumination and experimentally difficult to measure (Edwards and Baker, 1993). The CO, assimilation rate initially increased with increasing amounts of Rubisco and was saturating at intermediate Rubisco values (6 pmol m-'), presumably because the light intensity used in these experiments was subsaturating. More linear relationships between CO, assimilation rate and Rubisco content have been observed when measurements were made at higher irradiances (Fur- bank et al., 1996; von Caemmerer et al., 1997).

Absorptance of the Leaf

Leaf absorptance measurements are important for esti- mations of the quantum yield of the photosystems. Figure 1B shows up to a 7% decrease in light absorptance for the

A B I

I

t C C

I

- i PSll

G H

1165

90 9 :: z P

85 I s

U

; v

0.6 e

0.3

0.0

0.8 w

% U ?

0.4 ;b

2. o

- c -

0.0

1 .o e z e

0.5 2 \

0.0 ..

O 5 10 o 5 10 15 Rubisco sites (vmol m')

Figure 1. Dependence of photosynthetic parameters on the Rubisco content in leaves of different transgenic F. bidentis plants with reduced amounts of Rubisco (circles) and control plants (triangles). A, Assimilation rate plus Rd (in pmol m-' s-'); 6, absorptance (%); C, mPsII; D, QpsI; E, q, of PSll and redox state of QA of PSll (equal to 1 - q,, at high values QA is reduced, at low values QA is oxidized, F, was used instead of F,' for the calculation of qp); F, redox state of PSI in the steady state (A, closed symbols) and during a saturating flash (A,,,, open symbols); G, ratio of BPs, (closed symbols) or Q,,,, (open symbols) and Qc0, (= [A, + R,]/[absorbed lightl); H, ratio of @psl/

@psII. Measurements were made with 620 pmol quanta m-' sC1 irradiance and 370 wmol mo1-l CO, in 21% O,. The intercellular co, concentration was approximately 200 pmol mol-'. Leaf temperature was 25°C. Air RH was between 50 and 60%. Fluorescence measurements were made with a blue LED so that no PSI fluorescence disturbed the PSll measurements (see "Materials and Methods").

more severe transgenic plants, which had up to a 30% lower chlorophyll content compared with control plants, which had an average chlorophyll content of 640 2 80 pmol m-'. Decreases in leaf chlorophyll content were previously observed for severely affected Rubisco antisense transformants (Furbank et al., 1996). There was no difference in the relative amount of chlorophyll a to b in the transformants compared with the control plants (average ratio of chlorophyll a to b: 5.1 t- 0.5).

QPSI and QPSII

Unfortunately, it is not possible to discriminate between the optical signal coming from the bundle-sheath chloro-

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1166 Siebke et al. Plant Physiol. Vol. 115, 1997

t

plasts and that coming from the mesophyll chloroplasts. Our interpretation of these results does not attempt to assign fluorescence or absorbance signals to either compartment.

Similar to the CO, assimilation rate, the @‘psII and (DpsI were lower in plants with low Rubisco content compared with control plants (Fig. 1, C and D). As reported by Fur- bank et al. (1996), this was accompanied by an increased qN in the transformants (Fig. 1E). Presumably, an increased thylakoid proton gradient existed in the transformants, down-regulating PSII. The plastoquinone pool was also more reduced in the transformants with low Rubisco content, as evidenced by the increase in 1 - qp (Fig. 1E).

The BpsI is determined by the number of photons hitting a reduced PSI (Ievel of A of the absorbance signal) and the availability of electron acceptors (level of A,,, of the absorbance signal). A,,, remained fairly constant (Fig. lF), showing that the decreased amount of Rubisco had not caused a limitation of acceptors for PSI. The low apSI in the transformants was caused by a high level of oxidized PSI (high level of A, Fig. 1F). An oxidized P700 pool and a reduced plastoquinone pool shows that the Cyt b, complex was controlling the electron flow in these plants (Weis and Lechtenberg, 1989; Kirchhoff and Weis, 1995).

If the low Rubisco level caused an inhibition of CO, fixation without an appreciable down-regulation of the C, cycle, one would expect that the resulting higher ATP requirement per mole of CO, fixed should alter the relationship between DpSI and and the ato,. Figure 1G shows that more quanta were required by both photosystems per mole of CO, fixed. This also confirms the measurements of von Caemmerer et al. (1997), which showed that in these transformants leakage from the bundle sheath has increased. At 620 pmol quanta mp2 s-l the relationship between aPsI and @‘psII (Fig. 1H) did not markedly change, with only the severest transformant showing a small increase in the ratio. This suggests that both photosystems were involved in the production of the additional ATP used in the C, cycle.

Light-Response Curves of Photosynthetic Parameters in Leaves with Different Rubisco Contents

Figure 2A shows the light dependence of the CO, assimilation rate for a control plant and two transformants with different amounts of Rubisco. As already shown by Fur- bank et al. (1996), the initial slope of these curves remains unaffected by different Rubisco contents. At low light intensities the efficiency of light interception, not Rubisco content, limits photosynthetic flux. The maximal rate of photosynthesis reached was far lower in the transformants than in the control plants and the light intensity required to saturate photosynthesis was lower in transgenic plants with low Rubisco content.

The BrsI and (DpsI1 decreased with increasing light intensity (Fig. 2, E and F). At low light in the region of the initial slope of the CO, assimilation curve, a11 plants had a similar (DpsI and This is consistent with the hypothesis that the transformants have no increased CO, leakage at low light intensities. When light saturation was reached in the

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assimilation curve, the QpS, and (DrsII in the transformants decreased below that of the control plants.

C0,-Response Curves of Photosynthetic Parameters in Leaves with Different Rubisco Contents

Figure 3A shows the CO, dependence of the CO, assimilation rate for three different plants. As previously shown (von Caemmerer et al., 1997), the initial slope of this response depends primarily on the activity of the PEP carboxylase and was not affected by the difference in the amount of Rubisco. In the transformants the assimilation rate was already saturated by approximately 50 pmol/mol interceIlular CO, concentration, whereas in the control plant approximately 80 pmol/mol CO, was required. The light intensity sets the maximum ceiling for the rate of CO, fixation in the control plants, whereas in the transformants, this is determined by the amount of Rubisco present. The lower Rubisco activity in the transformants would lead to higher CO, concentrations in the bundle sheath at equal mesophyll intercellular CO, concentrations (von Caem- merer et al., 1997).

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Quantum Requirement of CO, Fixation in Transgenic Haveria bidentis 1167

-- 30 4 I

transformants

0.8

o

0.4

0.0

0.8 4

ir

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u

- o _,. _-,,.., I ,,,,, g.;L.r-----.. ................... ____._. -.-----.. Q

0.0 E 0.0

O 50 100 150 O 50 100 150 200

lntercellular CO, concentration (pmol mot )

Figure 3. CO, dependence of photosynthetic parameters in plants with different Rubisco contents. Measurements were made with two different transformants and a control plants having a Rubisco content of 2.3 (squares), 3.1 (circles), and 15 (triangles) pmol of Rubisco sites mz. The absorbed light was approximately 550 pmol m-’ s-’, differ- ing slightly between the plants at 530, 550, and 570 kmol m-’ s-’. The O, concentration was 21%. Relative air humidity increased with decreasing CO, concentration from 60 to 85%. Leaf temperature was between 24 and 25°C.

The regulation of the light reactions at low CO, concentrations is frequently comparable to that at high light intensities. In both conditions, excess energy has to be dissi- pated from PSI and PSII. Therefore, with decreasing CO,, qN, 1 - qF, and A increased in a manner similar (Fig. 3, B, C, and D) to that with increasing light intensity (Fig. 2, B, C, and D). These data also indicate that electron transport in the transformants was restricted by flux through the Cyt b, complex. Even at the lowest CO, concentrations A,,, (Fig. 3B) was fairly high. Accordingly, there appeared to be no limitation of electron acceptors for PSI. At low CO, concentrations the Qrs, (Fig. 3E) was similar in control plants and transformants. It is interesting that the curves of the QpsI against intercellular CO, concentration did not extrap- olate to O. This positive intercept could be the result of electron cycling around PSI either directly to P700+ as a futile cycle or via the Cyt b, complex, producing a proton gradient. Linear electron transport can play only a minor role since the curves of QrsII showed no significant positive intercepts at 2% O, (data not shown).

Estimated Leakiness and ATP and NADPH Requirements

We used a simple model to compare the measured quanta used per mole of CO, fixed with the requirements

estimated from the rates of CO, assimilation (”Appendix B”). We wanted to explore whether the measured quantum use of PSI and PSII could be used to assess the extent of leakiness in the transformants compared with controls.

From our gas-exchange measurements we can estimate maximal possible leakiness and use this to estimate quantum requirements per mole of CO, fixed in both control plants and antisense transformants (see “Appendix B”). In wild-type F. bidentis plants the leakage expressed as a fraction of CO, fixed by PEP carboxylase is approximately 0.24 (Hendersen et al., 1992; von Caemmerer et al., 1997). Therefore, a CO, fixation rate of 28 pmol m-’s-’ implies a PEP carboxylation rate of approximately 37 pmol mP2 s-’ in the control plants. If the capacity of the C, cycle is unchanged in antisense plants, a net CO, fixation rate of 14 pmol m-’ s-l in the most severely affected plants would imply an overcycling of 23 pmol m-, s-’ through the C, cycle, with a maximum estimate of leakiness of approximately 0.6. Figure 4A shows the maximal expected leakiness for leaves with different Rubisco contents calculated from measurements of the CO, assimilation rate (Fig. 1A). Any coordinate down-regulation of the C, cycle would reduce this value of leakiness.

From the estimated leakiness and the rate of CO, fixation, an ATP and NADPH requirement can be calculated (see “Appendix B”). Because of the inhibition in the Calvin cycle, the absolute energy requirement is less in the transformants than in the control plants (Fig. 48). The relationship between the ATP and NADPH requirements increased in transformants with reduced amounts of Rubisco. (Fig. 4C). Whereas the requirement for NADPH per mole of CO, fixed remained constant at 2 mol, the requirement for ATP per mole of CO, fixed increased from approximately 5.6 to about 8 mol.

Comparison of Estimated and Measured mPs, and a,,,, The exact quantification of the efficiency of energy con-

version from light to ATP is still controversial. The existence of a Q-cycle mechanism for electron transport through the Cyt b, complex has increasing support in the literature (Mitchell, 1977; Ort, 1986; Rich, 1988; Furbank et al., 1990; Heber et al., 1995; Kobayashi et al., 1995b). The ratio between H’ transfer and ATP production has been reported to be either 3 or 4 (Graber et al., 1987; Rumberg et al., 1990; Kobayashi et al., 1995a). Additionally, it is not known how ATP production is achieved without the production of NADPH, i.e. whether it is by cyclic electron transport from PSI to the Cyt b, complex, by linear electron transport to electron acceptors other than NADE‘+, or by both.

The different assumptions for the Q cycle and the H’/ ATP ratio change the relationship between the quantum requirement in the photosystems and the leakiness (Table I; ”Appendix B”; Furbank et al., 1990). We obtained reason- able fits between the theoretical curves and the data with the assumption that there is a Q cycle in the Cyt b, complex and that 4 Ht are transferred per ATP produced. Similar conclusions were reached by Gerst et al. (1994) for energy conversion in C, leaves. The lines in Figure 5 show the calculation of three different scenarios for the quanta re-

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O 2 4 6 8 1 0 1 2 1 4 Rubisco sites (pmol m-2)

Figure 4. Photosynthetic parameters calculated from gas-exchange data with the given assumptions. A, Maximal expected leakiness (VI), calculated from photosynthesis with the assumption that the PEP carboxylation rate remains the same in all plants (V , = 37 pmol m-' s-') and V, was calculated from the assimilation rate of the control plants with the known leakiness (VIcOnfr0, plant = 0.24, von Caem- merer et al., 1997). B, ATP and NADPH requirement (pmol m-* s-'), as calculated from the maximal expected leakiness. C, Ratio of the ATP/NADPH requirement, as calculated from the maximal expected leakiness.

quired by PSI and PSII per CO, fixed with changing leakiness. In the first scenario (Fig. 5A) we assumed that a11 extra ATP is produced via linear electron transport to alternative electron acceptors; in the next scenario, a11 extra ATP is produced via cyclic electron transport from PSI to the Cyt b, complex (Fig. 58); and in the last scenario, one-half of the extra ATP is produced via cyclic electron transport, and the other half is produced via linear electron transport.

To compare the theoretical relationship of leakiness and the quantum requirement of PSII and PSI with the measured results, we have plotted the measured quanta used by PSI and PSII per CO, fixed versus the estimated leakiness (symbols in Fig. 5). The determination of quantum yield depends on the fraction of light absorbed by the photosystems and, unfortunately, it is not known at what

ratio the absorbed light was distributed between PSI and PSII. However, from the unchanged ratio of chlorophyll a to b it can be concluded that the relative antenna size of PSI and PSII was not affected by the transformation. We have neglected the possibility that state 1 / state 2 transitions may have changed the light distribution and could be different between controls and transformants (Weis, 1985; Williams and Allen, 1987; Horton and Hague, 1988).

Three different assumptions for the light distribution were made for the different scenarios shown in Figure 5. A comparison of the position of data points in Figure 5 dem- onstrates the effect that partitioning of light between PSI and PSII has on the calculated quantum use, In Figure 5A the light distribution between PSI and PSII was chosen to give the same quantum yield for both photosystems. This would be necessary if the ATP required in addition to that produced during obligatory NADPH production were to be generated by linear electron transport, because in this case, every electron has to pass through both photosystems. In Figure 5B the light distribution was chosen to fit the theoretical relationship between leakiness and quantum requirement, if a11 of the extra ATP is provided via cyclic electron transport from PSI to the Cyt bf complex. In this case, the aPsII should not change with leakiness. In Figure 5C we assume that 50% linear electron transport to alternative electron acceptors and 50% cyclic electron transport are used to generate the extra ATP.

The best fit is achieved with the assumptions made for Figure 5C. However, the question of whether the extra ATP (ATP not supplied during linear electron transport to NADPH) is provided preferentially by cyclic or linear electron transport cannot be answered conclusively. The increase in the QFsII with increasing leakiness (Fig. 58) indicates that linear electron transport per net CO, fixed increased with leakiness, not just cyclic electron transport. The fit in Figure 5A required the assumption that PSI absorbed less of the light than PSII, which seems unlikely for a C, plant and does not fit with our data obtained at very low CO, concentrations, where the ratio of PSI to PSII decreases to 0.75 (Fig. 7; "Discussion").

As the points were positioned at their maximal expected leakiness, their closeness to the curve shows that there was no appreciable down-regulation of the C, cycle activity in the transformants; otherwise the measured quanta used per CO, for the transformants would have stayed below the theoretical line. This differs from . the observations of von Caemmerer et al. (1997) using A13C measurements made on the same plants at high irradiance. They found an incomplete down-regulation of the C, cycle resulting in a lower leakiness in the transformants (1Ir = 0.37) than the maximal expected.

Plants used here had similar Rubisco contents as the plants used by von Caemmerer et al. (1997) and lay on the theoretical curve with values of approximately 0.42 in our experiments. It is possible that down-regulation of the C, cycle occurs mainly at high irradiance, which may explain the decrease in the measured quantum use under these conditions (Fig. 6). Another factor that could contribute to the difference between the two estimates would be any additional ATP-consuming processes that do not change

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Quantum Requirement of CO, Fixation in Transgenic Flaveria bidentis 1169

Table 1. Enerw consumption for CO, fixation

Ratios Variables and Equations

ATP per CO, fixed

NADPH per CO, fixed Quanta used per 2 NADPH

By PSll By PSI

2 3 f - 1-?

2

4 4

Q-cycle No Q-cycle Linear electron transport

PSll quanta/H+ 1 / 3 1 /2 b PSI quanta/H+ 113 1 /2 b

PSI quanta/H+ 1 12 1 C

Cyclic electron transport around PSI

Quanta used per ATP, if produced by linear electron transport

By PSll By PSI

Quanta used per ATP, if produced by cyclic electron transport

By PSI

ATP produced per 2 NADPH

Extra ATP required per mo1 of CO, fixed

H+ H+ H+ 4- ATP 3- ATE' 3- ATP

H+ ATP 4- a

1 1.33 1.5 1 1.33 1.5

1.5 2 3

4 3 2.66

2 2 1 2 ~

5 + F F -l+- 1 - W 1 - W

2 N, = a . b 2 N, = a . b

4 N, = a . c

2 4 Ni -

4 A =d-- * Ni

2 1 +- 1-9

A13C values but increase the ATP requirement per mole of CO, fixed. Such a process has recently been reported; PEP carboxylase dephosphorylated about 5% of PEP without achieving a carboxylation (Scott et al., 1992).

The Quantum Requirement of CO, Fixation at Different lrradiances

We have used the assumptions of Figure 5C to compare the measured and estimated quantum requirement at different irradiances. Figure 6 shows that the quanta used per CO, fixed in PSI and PSII and their ratio remained rela- tively constant across irradiance in the control plants. This is consistent with previous observations that leakiness does not vary with irradiance except perhaps at very low light (Krall et al., 1991; Henderson et al., 1992). CO, assimilation rates of control and transformant plants were similar at low light intensities and diverged only at higher light intensities, because the low Rubisco contents led to the early saturation of the light-response curves (Fig. 2A). The aPsI and QPsII and their ratio were also similar for control and transformants at low light (Fig. 6).

The ratio of aPsI to aPsII increased at higher light intensities in a manner similar to the estimated ATP-to-NADPH ratio (Fig. 6, A and D). An increase of aPsI/ aPsII is always an indication that cyclic electron transport occurs, either to the Cyt b, complex to produce ATP or as a futile cycle directly to P700+ to deplete energy. A more quantitative comparison is given in Figure 6, which shows the quanta

actually used to fix one CO, (Fig. 6, B and C) and the estimated quantum requirement per net CO, fixed (Fig. 6, E and F). The quanta used by PSII per CO, fixed (Fig. 6B) increased more steeply with increasing light intensity than predicted and, surprisingly, it declined at higher light intensities. Perhaps this indicates some down-regulation of the C, cycle. In contrast, quanta used in PSI in the high light region (Fig. 6C) are more than expected (Fig. 6F). It appears that the role of cyclic electron transport is more important in high light intensities than was assumed in Figure 5C.

lnfluence of Photorespiration on the Quantum Requirement of CO, Fixation

We have so far assumed that bundle-sheath CO, concentrations are sufficiently high that under atmospheric CO, concentrations, photorespiration is not a significant sink for electrons. Figure 7 shows the quantum requirement at different CO, concentrations. The amount of quanta used by PSI and PSII increased at very low CO, concentrations. This was presumably due to an increase in RuBP oxygenation, since the bundle-sheath CO, concentration at the compensation point is at the C, compensation point (approximately 50 pbar). Similar curves for PSII have previously been observed in different C,, C,-C,, and C,-like Flaveria sp. by Krall et al. (1991).

The higher rates of RuBP oxygenation in the control plants compared with the transgenic plants led to a cross-

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11 70 Siebke et al. Plant Physiol. Vol. 11 5, 1997

O

. . . . . . only linear

e- transport

' / $ ..

light distribution: PSI: 467'0, PSII: 54% A

I , , ,

e- cycling around PSI /

PSI: 62%, PSII: 38% B , ,

50% linear 50% cycling

0.0 0.2 0.4 0.6

leakiness

Figure 5. The theoretical estimates of the quantum requirement of PSI and PSll for CO, fixed as a function of leakiness (solid line, PSI; dashed line, PSII) are compared with the calculated number of quanta used by PSI and PSll from experimental data shown in Figure 1. Solid symbols, PSI; open symbols, PSII; triangles, control plants; circles, different transformants. For the theoretical estimates it was assumed that there is a strict Q cycle in the Cyt b, complex and that four protons are required per ATP produced (see "Appendix B"; Table I). The horizontal position of the data points was determined from gas-exchange measurements of maximal expected leakiness (compare Figs. I A and 4A). The vertical position was determined from optical measurements (Fig. 1G) in combination with different assumptions for the light distribution between PSI and PSII, chosen to fit with the theoretical curves. Three possible scenarios are shown: A, Extra ATP (not produced by the obligatory linear electron transport to NADPH) is produced via linear electron transport to alternative electron acceptors (light distribution was assumed: 46% is absorbed by PSI and 54% by PSII); B, extra ATP is produced only by cyclic electron transport from PSI to Cyt b, complex (light distribution was assumed to be 62% absorbed by PSI and 38% absorbed by PSII); and C, extra ATP is produced by 50% cyclic electron transport from PSI and 50% linear electron transport to alternative electron acceptors (light distribution was assumed to be 57% absorbed by PSI and 43% absorbed by PSII).

over of the curves for PSII (Fig. 7B) for the transformants and control plants. It is likely that the bundle-sheath CO, concentration in the transformants increases more rapidly from the compensation point value because of the reduced amounts of Rubisco. We examined this in more detail with a comparison of measurements made at 2 and 21% O, (Fig. 8). In the control plant the quanta used by PSII at low CO, was lower in 2% compared with 21% O,, which is consistent with a reduction in photorespiration. This difference is less marked in the transformants (Fig. 8B). The quanta used by PSI per CO, is not influenced by O*. Its increase in low CO, concentrations may also be due to a futile cycling of electrons to oxidized PSI, which masks the effect of O,.

For each oxygenation of RuBP, four redox equivalents (equivalent to 2 NADPH) and 3.5 ATP are required (Far- quhar and von Caemmerer, 1982). We cannot quantify to what extent the required ATP and redox equivalents are

. . - anti-Rubisco . ..... . * a - . - . .:

control

c 1 ' F 1 5 O 500 1000 1500 O 500 1000 1500 2000

absorbed light ( p o l quanta m-2 s-1)

Figure 6. Comparison of the measured quantum yield of PSI and PSll from optical measurements (left) with the estimated quantum requirement from gas exchange (right). Data are from Figure 2. Calculations were done with the same assumptions as in Figure 5C. A, Ratio of @.psI/@ps,I. B, Quanta used by PSll per mole of CO, fixed calculated from the data in Figure 2 as 0.43 @psl,/@coz, where 0.43 i s the fraction of light absorbed by PSII. C, Quanta used by PSI per mole of CO, fixed, calculated from the data in Figure 2 as 0.57 @)PS,/@CO,,

where 0.57 is the fraction of light absorbed by PSI. D, Estimated ratio of ATP and NADPH requirement calculated from the maximal expected leakiness. It was assumed that leakiness increases with increasing light intensity in the transformants while it remains constant in the control plants (~transfor,,,anIs = 1 - [(Ac + Rd)transformants . (1-0.24)/(Ac + Rd)conIroll at each irradiance). From the leakiness and CO, assimilation rate, the ATP and NADPH requirement was calculated according to Table I and "Appendix B." E, Estimated quantum requirement of PSll calculated from the ATP and NADPH requirement with maximal expected leakiness (Table I; "Appendix B"). F, Estimated quantum requirement of PSI calculated in the same way (Table I; "Appendix B").

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Quantum Requirement of CO, Fixation in Transgenic Flaveria bidentis 1171

& transformants ...-....... _.... .-..-------- O

control -1

O 50 100 150 200 lntercellular C02 Concentration

(pmol/mol)

Figure 7. Comparison of mPsI, @.p511, and Qe,, at different CO, concentrations. A, @psI/QpsII; B, quanta used by PSll per CO, fixed; C, quanta used by PSI per CO, fixed. Data are from Figure 3. Calculations were as described in Figure 6, A to C.

produced in the mitochondria and how this influences the balance of NADPH and ATP production in the chloroplasts. Nevertheless, Figure 7A shows that the ratio aPslI/ aPs1 decreased in the control plants in the region of high photorespiration, suggesting that there was some cyclic electron transport in higher CO, concentrations, which decreased when photorespiration increased. Obviously, the ATP/ NADPH requirement in the chloroplasts decreased under photorespiratory conditions. In contrast to the control plants, the transformants showed no decrease of cyclic electron transport in low CO, concentrations, indicating that they apparently carried out less photorespiration. With very low CO, concentrations, increased in the transformants, and in the control plant this might have been due to a futile cycle of electrons to oxidized PSI.

A conclusion about the light distribution between PSI and PSII can be drawn from the minimum of the curve for

the control plant in Figure 7A. If the light distribution between PSI and PSII was equal, then / aPsII should have resulted in values greater than or equal to 1, since every electron that passes through PSII must also pass through PSI. The ratio of ~ p s I / ~ p s I I shown in Figure 7A reached values of less than 0.75, which implies that at least 57% of the light was absorbed by PSI, and 43% was absorbed by PSII. This distribution coefficient is the same as the one assumed for Figure 5C.

A

I A \ B i

,

O 10 20 30 40 50 lntercellular CO concentration

(pmol/mol) 2

Figure 8. lnfluence of O, on the C 0 2 assimilation rate (A) and on the number of quanta used in photosystems I I (6) and I (C) to fix 1 mo1 of CO,. Measurements were done for a control plant: open (2% O,) and closed (21% O,) triangles, and a transgenic plant: open (2% O,) and closed (21% O,) squares by varying the O, concentration at each CO, concentration. Light intensity was 620 pmol quanta m-'s-', and leaf temperature was 25OC. The Rubisco site concentration was 12.6 and 1.9 pmol m-' and the absorbed light was 542 and 51 2 pmol quanta m-, s - l for control and transgenic plants, respectively. Quantum use was calculated as described in Figure 6.

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CONCLUSION

We have confirmed with optical measurements that decreased Rubisco content leads to increased CO, concentration in the bundle sheath and to increased leakiness. Al- though uncertainties of light absorption, Q-cycle activity, and proton requirements of ATP production make it difficult to achieve quantitative estimates of leakiness, we were able to show that the higher bundle-sheath CO, concentrations in transformants suppress photorespiration at very low CO, concentrations. We used a model to compare measured and estimated quantum requirements and concluded that the increased ATP / NADPH requirement in the transformants led to an increase of quanta used per mole of CO, fixed in both photosystems and that cyclic electron transport around PSI may be used for at least one-half of the generation of extra ATP for the C, cycle. However, it is also clear that not a11 of the extra ATP is generated solely by cyclic electron transport, since linear electron transport per mole of CO, fixed also increased substantially in the transformants.

APPENDIX A: PROCEDURE FOR MEASURINC A,,,

The level of A,,, determines the amount of PSI centers unable to perform photochemistry because of the full reduction of electron carriers on the acceptor side of PSI. In a dark-adapted leaf a11 PSI have an oxidized acceptor side and it is possible to fully oxidize PSI to the level of A,,,. Figure 9A shows the procedure for measuring A,,,. After 20 s of far-red illumination P700 is fully oxidized by a

A B

fast-saturating white-light pulse (Klughammer and Schrei- ber, 1994). Figure 9B shows the procedure for measuring A,,,, P700 oxidation during actinic light, as suggested by Klughammer and Schreiber (1994). During the measurement, a saturating light flash is given to oxidize a11 P700 except the photosystems with a reduced acceptor side. The absorbance of P700+ reaches A,,,. Varying from the procedure suggested by Klughammer and Schreiber (1994), we gave a short (0.5 s) flash of far-red light before the saturating white light (Fig. 9C). The far-red light depletes electrons from the electron transport chain, and the subsequent fast white flash oxidizes PSI to A,,,. Without the short far-red light pulse, the re-reduction of P700+ during the saturating white pulse is so fast that it is difficult to extrap- olate to A,,,, especially in low actinic light intensities. It should be noted that our saturating white-light pulse was of lower intensity than the one recommended by Klugham- mer and Schreiber (1994). The described procedure arose from the observation that in a dark-adapted leaf it is not possible to fully oxidize P700 with a saturating white-light pulse no matter how high, without giving the far-red light first (C. Klughammer, personal communication).

Theoretically, the far-red light used here might decrease the amount of available acceptors for PSI and therefore decrease the obtained A,,,. However, the values obtained for A,,, were up to 25% higher in low actinic light intensities (250 pmol mp2 s-l) and up to 3% higher in high actinic light intensities (2400 pmol m-* s-l) with the short far-red light pulse than without it.

C

t i

Figure 9. Procedure to measure the P700f absorbance levels required for the calculation oí A, Example for a leaf in the dark-adapted state, performed to determine A,,, and A,. Far-red light (FR) is given for 20 s (1 20 pmol íar-red light m-* s-') to deplete electrons írom the electron transport chain (the last second i s shown). With a saturating flash oí white light (9500 pmol m-* s-') P700 becomes fully oxidized, resulting in an absorbance level oí A,,,. In the following dark period P700f becomes fully reduced and i ts absorbance reaches A,. B, Procedure suggested by Klughammer and Schreiber (1 994) to determine A, A,,,, and A,. A saturating flash of white light is given to oxidize P700 to the level of Asat. In the following dark period the A, can be determined. C, Procedure used to determine A, A,,,, and A,. The measurement was períormed as described in B, except that a 0.5-s pulse of far-red light is given in addition to the actinic light before the saturating white-light pulse. A higher level of A,,, is reached (see text).

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Quantum Requirement of CO, Fixation in Transgenic Flaveria bidentis 11 73

APPENDIX B: MODEL TO CALCULATE THE QUANTUM REQUIREMENT OF PSI A N D PSll PER MOLE OF CO, FIXED

To link CO, fixation to the QpsI a n d QpsII, the ATP and NADPH requirement of CO, fixation needs to be known. W e can ignore photorespiration (except a t very low light a n d CO,) and

A, + Rd = V, = Vp - L, (1)

where V, and Vp are the rates of RuBP a n d PEP carboxylation, respectively, L is the CO, leak rate out of the bundle sheath, and A, denotes the net CO, assimilation.

Leakiness, V, is defined as

and

Vp = (A, + R J / ( l - T). ( 3 )

ATP is required i n the C, and the C, cycle, and the overall demand is

ATP requirement = (A, + Rd) (4)

NADPH is required only i n the C, cycle a t the rate of 2V, (Table I) and 4 quanta are required for the production of one NADPH (two i n each photosystem). The number of quanta required to produce one ATP depends on the exis- tente of a Q cycle i n the Cyt bf complex and on the number of protons transferred per ATP produced (Table I). These parameters also influence how much ATP can be produced together with NADPH. Table I shows the results for the different assumptions. Extra ATP has to be produced i n addition to that produced by the obligatory linear electron transport for the C, cycle and, in absence of a Q cycle, also for the C, cycle. The extra ATP can be produced either by linear electron transport to alternative electron acceptors or by cyclic electron transport a round PSI. Since the relative importance of these t w o options is unknown, we have introduced the variable m and O I m 5 1.

Linear electron transport to alternative e- acceptors Linear e- transport to alternative e- acceptors

+ cyclic e- transport around PSI ( 5 )

m = ( The quanta used by PSII per CO, fixed are the sum of the

quanta used for the production of NADPH plus the quanta used for the extra ATP (Table I):

1

Quanta used by PSII per C02 = 4 + m * A, N, (6)

where N, denotes quanta used per ATP in linear electron transport and A, denotes the extra ATP, which is ATP required per CO, fixed in excess of that produced via the reduction of NADPH. (Table I). The same amount of quanta are used b y PSI per CO, fixed i n the linear electron transport. In addition, PSI might use quanta in the cyclic

electron transport to produce extra ATP:

Quanta used by PSI per COz =

4 + WI A, * N, + (1 - m) * A, * N, (7)

where N, denotes quanta used per ATP i n cyclic electron transport (Table I). We have assumed the operation of a Q cycle in the Cyt b, complex a n d that there are four protons transferred per ATP produced. In this case the equations simplify to:

PSII quanta per COz = 4 + m * 2.66/ (1 - V) (8)

and

ACKNOWLEDCMENTS

We wish to thank Sari Ruuska for her help in introducing K.S. to the lab and particularly for her instruction in Rubisco measurements. We thank Christof Klughammer for interesting discussions and helpful suggestions about the P700 measurements and An- thony Millegate for help with plant culture.

Received April 17, 1997; accepted July 18, 1997. Copyright Clearance Center: 0032-0889/97/ 115/1163/ 12.

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