Decreased ribulose-l,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with 'antisense' rbcS. VI. Effect on photosynthesis in plants ...
Planta (1993)190:332-345
P l a n t a 9 Springer-Verlag1993
Decreased ribulose-l,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with 'antisense' rbcS VI. Effect on photosynthesis in plants grown at different irradiance M. Lauerer ~, D. Saftic 2., W.P. Quick 2.*, C. Labate 2.**, K. Fichtner 1, E.-D. Schulze 1, S.R. Rodermel 3, L. Bogorad 3, M. Stitt 2.*** 1 Lehrstuhl ftir Pflanzen6kologie, Universitfit Bayreuth, Postfach 10 12 51, W-8580 Bayreuth 2 Lehrstuhl ffir Pflanzenphysiologie,Universit/it Bayreuth, Postfach 10 12 51, W-8580 Bayreuth 3The Biological Laboratories, Harvard University, Cambridge, MA 02138, USA Received: 9 September 1992/ Accepted: 30 December 1992
Abstract. Tobacco (Nicotiana tabacum L.) plants transformed with 'antisense' rbcS to decrease the expression of ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO2 at 20~ at 100, 300 and 750 g m o l . m 2.s-1 irradiance, and at 28~ at 100, 300 and 1000 Dmol.m 2. s-I irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (CRubisco) A was very low (0.01-0.03) at low growth irradiance, and still fairly low (0.24-0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that CRubisc A o increases as incident irradiance rises. (iii) When low-light (100 Dmol'm 2's 1)-grown plants are exposed to high (750-1000 Dmol-m 2"s ~) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750 1000 ~tmol.m 2.s t) are suddenly exposed to high and saturating irradiance (1500-2000Dmol.m-2-s 1), CRubisco A remained relatively low (0.23-0.33), showing that in saturating light Rubisco only exerts partial control over the * P r e s e n t a d d r e s s : Institute of Field and Vegetable Crops, University of Novi-Sad, 21000 Novi-Sad, Serbia ** P r e s e n t a d d r e s s : Department of Molecular Biology and Biotecbnology, University of Sheffield, U K *** P r e s e n t a d d r e s s : Department of Biology, Queens University, Kingston, Canada **** P r e s e n t a d d r e s s : Botanisches Institut, Im Neuenheimer Feld 360, W-6900 Heidelberg
Abbreviations: A = rate of photosynthesis; CRubiscoA = flux control coefficient of Rubisco for photosynthesis; c~= internal CO2 concentration; qE=energy-dependent quenching of chlorophyll fluorescense; qQ = photochemical quenching of chlorophyll fluorescence; N A D P - M D H =NADP-dependent malate dehydrogenase; Rubisco=ribulose-!,5-bisphosphate carboxylase-oxygenase; RuBP= ribulose- 1,5-bisphosphate C o r r e s p o n d e n c e to:
M. Stitt; FAX: 49(6221)5658 59
light-saturated rate of photosynthesis in " s u n " leaves; apparently additional factors are co-limiting photosynthetic performance. (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the "light" reactions and Rubisco; this was shown by the low value of CRubisc o A (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions. (v)'Antisense' plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.
Key words: Light climate - Nicotiana (photosynthesis) Photosynthesis - Ribulose-l,5-bisphosphate carboxylase-oxygenase - Transgenic plant (tobacco, antisense DNA)
M. Lauerer et al. : Decreased Rubisco in transformed tobacco. VI Introduction
The following experiments were carried out to investigate whether ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) limits the ambient rate of photosynthesis in tobacco plants grown in a range of different irradiances and temperatures. Ribulose-l,5-bisphosphate carboxylase-oxygenase is often considered to play a cardinal role in controlling the rate of photosynthesis. The enzyme has a rather low catalytic activity (Andrews and Lorimer 1987; Woodrow and Berry 1988) and the net rate of carboxylation is further decreased by its low affinity for COz and by the competing oxygenase reaction (Andrews and Lorimer 1987). This inefficiency is partially compensated by the large amount of Rubisco in the leaf (corresponding to 20-25% of total leaf protein, Evans 1989), and by the high concentration of ribulose-l,5-bisphosphate (RuBP) in the leaf during rapid photosynthesis (Woodrow and Berry 1988; Sharkey 1989). Most previous studies of the control of photosynthesis have used sophisticated mechanistic models (e.g. Farquhar and von Caemmerer 1982; Sharkey 1985; Sage 1990) to interpret measurements of gas exchange, enzyme activities and metabolites (e.g. von Caemmerer and Edmondson 1986; Sage et al. 1988; Sharkey 1989; Sage et al. 1990). These models assume that photosynthesis is limited by single factors and that all other components will be in excess, and are down-regulated to match the single limiting step. On the basis of this approach, it has been proposed that Rubisco limits the rate of photosynthesis in air at saturating irradiance, and also in low C Q . However, it has been suggested that the maximum rate of photosynthesis can be restricted by stomatal conductance (Farquhar and Wong 1984; Jones 1985; Woodrow et al. 1990) or resistance to COz diffusion through the aqueous phase of the leaf (Evans and Terashima 1988). The light-saturated rate of photosynthesis can also be limited by the rate of sucrose and starch synthesis at low temperatures (Leegood and Furbank 1988) or high CO2 (Sharkey 1989; Stitt and Quick 1989). The study of the control of photosynthetic rate is further complicated by the long-term adaption of the photosynthetic apparature to high or low light. This phenomenon has been investigated by growing plants at different irradiances and studying the effect on photosynthetic responses to light or CO/, as well as the composition of leaves. Leaves which have developed in low light ("shade" leaves) usually have a lower light-saturated rate of photosynthesis than leaves which have developed in high light ("sun" leaves), whereas the rate of photosynthesis at low measuring irradiance is similar or even higher (Bj6rkman 1981). "Shade" leaves have a decreased in-vitro ratio of Rubisco activity / Hill reaction activity (Terashima and Evans 1988), an increased chlorophyll (Chl) content relative to the electron-transport components (Leong and Anderson 1986; Evans 1987; Terashima and Evans 1988) and a decreased Chl a/b ratio (indicative of increased light-harvesting complex; Leong and Anderson 1984; Evans 1987). It has been argued that these changes represent an adjustment to low irradiance, in
333 that they allow nitrogen resources to be reallocated to maximise light interception and harvesting (Field 1983). In agreement, analysis of curves of rate of photosynthesis (A) versus internal CO2 concentration (cl) indicate that the balance between Rubisco and RuBP regeneration is maintained across a range of growth irradiances (von Caemmerer and Farquhar 1981; Evans 1987). However, Lee and Whitmarsh (1989) did not find significant changes in the composition of pea thylakoids in low-irradiancegrown plants, and argue that the changes reported by other groups are anyway too small to represent an effective adaption to large alterations in the growth irradiance. A more direct way to measure the contribution of Rubisco to the control of photosynthesis is to use tobacco plants which have been transformed with 'antisense' rbcS (the gene for the nuclear-encoded small subunit of Rubisco, Rodermel et al. 1988) to experimentally determine the flux control coefficient of Rubisco for photosynthesis (CRubisco)" A The flux control coefficient is defined (Kacser and Porteous 1987) as C -
dJ/J dE/E
(Eq. 1)
where dJ/J is the fractional change in steady-state flux through the pathway which is produced by a fractional decrease dE/E of the amount of one enzyme (leaving all other enzymes unaltered). The flux control coefficient can be evaluated from the slope of a normalised plot of the rate of photosynthesis versus Rubisco content for a series of genetically manipulated plants provided (i) that the amounts of other proteins have not been changed (see Quick et al. 1991a) and (ii) that only a relatively small part of the wild-type enzyme is removed in the first plant of the series (strictly speaking, the flux control coefficient is a partial differential and its value usually rises as an increasingly large part of the normal enzyme is removed, see Kruckeberg et al. 1989, also Ouick et al. 1991a). In interpreting the relative importance of individual components it is helpful to note that the sum of all the coefficients for a given system is usually unity (see Kacser and Porteous 1987 for a more detailed discussion). When tobacco plants were grown at high nitrogen supply and an irradiance of 340 gmol 9m - z. s- 1, 'antisense' rbcS plants with 43% less Rubisco than the wild type showed only a marginal (7%) inhibition of photosynthesis, demonstrating that Rubisco was only exerting a small amount of control (CRubisco a ~ 0.1, Quick et al. 1991a, b). However, we also found that short-term changes in the irradiance dramatically altered the extent to which Rubisco controlled the rate of photosynthesis (Stitt et al. 1991). At a measuring irradiance of 1000 tamol" m -2" s -1, Rubisco was the major control site of photosynthesis (CRAublsco~0.8). The following experiments were therefore carried out to investigate whether plants which have grown at high light intensities retain a Rubisco-limitation of ambient photosynthesis, or whether acclimation reestablishes a balance between the various components of the photosynthetic apparatus such that Rubisco again only
334
M. Lauerer et al.: Decreased Rubisco in transformed tobacco. VI
exerts marginal control over the rate of photosynthesis in g r o w t h c o n d i t i o n s .
after Hall and Totbert (1978) and its purity checked by denaturating sodium dodecyl sulfate (SDS)-gel electrophoresis. To quantify protein content in the pure Rubisco, absorption was measured at 280 nm and protein content was calculated as in Paulsen and Lane (1966). In an earlier publication we quantified Rubisco in tobacco leaves by rocket immunoelectrophoresis (Quick et al. 1991a). The amounts of Rubisco protein detected in the ELISA test used in the present article are five-fold lower than those we reported previously, because our earlier measurement were an overestimation due to impurities in the Rubisco standard and errors in quantifying protein in the standard. To measure soluble and insoluble protein, leaf discs (4 c m 2) w e r e homogenised in 1 ml of solution containing 50 mM 4-(2-hydroxyethyl)-l-piperazine ethanesulfanic acid (Hepes)-KOH (pH 7.9), 5 mM MgCI2, 5 mM EDTA, 10% (v/v) glycerol and 5 mM dithiothreitol, centrifuged (14000-9, 20 min) and soluble protein determined in the supernatant as in Bradford (1976). The sediment was washed with buffer, dissolved in 1 ml 10% trichloroacetic acid, centrifuged (14000.9, 20 min) and the sediment then dissolved in 1 ml 0.1 M NaOH, centrifuged (14000.9, 20 min) and insoluble protein then measured in the supernatant. The trichloroacetic-acid step removes chlorophyll, which interferes with the protein assay. In preliminary experiments, pellet was also solubilised with 1% (v/v) Triton X-100 and 1% (v/v) SDS, diluted to a concentration where detergent did not interfere with the standard assay, assayed as in Bradford (1976), and corrected for interference due to chlorophyll by carrying out a control in which a similar concentration of chlorophyll was added directly to the protein assay. Similar results were obtained with Triton X 100, SDS and trichloroacetic acid/ NaOH extraction.
Material and methods The selfed progeny of transformants 3 and 5 of tobacco (Nicotiana tabacum L. SRI, Rodermel et al. 1988) were used in these studies. The 'antisense' line "3" contained one copy of 'antisense'; selfing generates pseudowild types, and two groups of plants with decreased Rubisco (approx. 40% and 60% reduction in a 1:2:1 ratio, see Quick et al. 1991a). The 'antisense' line "5", which contained several copies of 'antisense' (Rodermel et al. 1988), had subsequently been selfed and progeny selected which were phenotypically small. These plants were again selfed to generate seed for the following experiments producing a set of reproducibly small plants. Seeds were germinated on moist filter paper at 20 ~ C, transferred to 1-1 pots filled with washed sand and grown at 20 ~ C or 28 ~ C, at about 75% humidity and irradiance between 100 and 1000 lamol 9m -z 9s- 1, as specified in the results. The irradiance was regulated by using green netting, and was checked over the entire surface at the horizontal level occupied by the plants using Quantum sensor (Lambda LI-185; LI-COR, Lincoln, Neb., USA). Plants were watered daily with 0.4 1 nutrient solution (containing 10 mM NH4NO3, 3 mM K2HPO4, 2 mM MgSO4, 2 mM CaSO4, 50 laM KC1, 25 [aM H3BOa, 20 laM Na-Fe-EDTA, 2 laM MnSO4, 2 laM ZnSO4, 0.5 laM CuSO4 and 0.5 gM MoO3 ; pH 6.0) per plant. Plants grown at 20 ~ C in 100, 300 or 750 gmol 9m 2. s 1 light irradiance were used after 41, 37 or 44 d of growth. All the plants grown at 28 ~ C were used after 33 d of growth. All measurements took place when the plants were still in a rosette-type growth stage and before the stem elongation and flowering commenced. Gas exchange was measured as previously described (Quick et at. 1991a) using a portable porometer (Zimmermann et al. 1988) connected to a remote leaf chamber located inside the growth room. The environmental conditions of the leaf inside the measuring cuvette were maintained as in the growth room. In some measurements the irradiance was decreased using neutral grey filters or was supplemented by a standard light projector. Carbon dioxide and H20 were detected continuosly by an infrared gas analyser (BINOS; Leybold HerS.us, Hanau, FRG). Assimilation of CO2, stomatal conductance to water vapor, transpiration and the internal CO2 partial pressure of the leaf were calculated as in van Caemmerer and Farquhar (1981). Chlorophyll flourescence was measured in the gas-exchange system using a PAM-fluorometer (Heinz Walz, Effeltrich, F R G ; as in Quick et al. 1991a). Malate dehydrogenase (NADP-dependent; N A D P M D H ) was extracted and assayed exactly as in Scheibe and Stitt (1988). Extracts for Rubisco determination were prepared in the light from material which had been illuminated for at least 9 10 h (to avoid possible complications due to carboxyarabitinol-1phosphate), and using the area of leaf previously used for the gas exchange measurement. Extraction and radiometric assay of Rubisco after preincubation with high Mg z + and CO2 at pH 8.0 to fully activate the enzyme were as described previously (Quick et al. 1991a). The amount of Rubisco was determined by enzyme-linked immunosorbent assay (ELISA) as described in Catt and Millard (1988; the plates were washed ten times with tap water instead of three times with different buffer solutions) using purified tobacco leaf Rubisco as standard. The reliability of the ELISA was checked with pure Rubisco in the presence of extracts. The slope was within 10% of the slope when standard alone was used. Polyclonal rabbit antiserum, raised to Medicago sativa Rubisco was used as in Cart and Millard (1988). The antibody was characterized by western blotting against an extract from tobacco leaves and against purified tobaccoRubisco ; only one band was found in both cases, indicating that the antibody was specific. The Rubisco had previously been purified
Results Growth and photosynthesis at 20 ~ C. T h r e e sets o f p l a n t s w e r e g r o w n at 100, 300 o r 750 ~tmol. m - 2 . s -1 irrad i a n c e a n d 20 ~ C. E a c h g r o u p o f p l a n t s c o m p r i s e d 30 60 i n d i v i d u a l s , i n c l u d i n g w i l d types, a n d s e g r e g a n t s f r o m a selfing o f line " 3 " , a n d s m a l l p r o g e n y f r o m t h e line " 5 " ' a n t i s e n s e ' p l a n t s ( R o d e r m e l et al. 1988). A f t e r m e a s u r ing p h o t o s y n t h e s i s , the p l a n t s w e r e h a r v e s t e d f o r d e t e r m i n a t i o n o f R u b i s c o , a n d s t r u c t u r a l analysis. T o m e a sure t h e a m o u n t o f R u b i s c o in the v a r i o u s g e n o t y p e s , w e (i) a s s a y e d m a x i m u m a c t i v i t y a f t e r p r e i n c u b a t i o n in the e x t r a c t s to fully a c t i v a t e R u b i s c o a n d (ii) d e t e r m i n e d Rubisco protein by ELISA. There was a direct linear r e l a t i o n b e t w e e n the R u b i s c o a c t i v i t y a n d t h e a m o u n t o f R u b i s c o p r o t e i n d e t e r m i n e d i m m u n o l o g i c a l l y f o r all irrad i a n c e s a n d g e n o t y p e s ( d a t a n o t s h o w n , r 2 = 0 . 9 6 , see also Q u i c k et al. 1991a; n o t e h o w e v e r t h a t o u r specific a c t i v i t i e s a r e n o w f i v e f o l d h i g h e r d u e to i m p r o v e m e n t s in the a b s o l u t e q u a n t i f i c a t i o n o f t h e E L I S A ) . R i b u l o s e - l , 5 b i s p h o s p h a t e c a r b o x y l a s e - o x y g e n a s e is e x p r e s s e d in t e r m s o f m a x i m u m a c t i v i t y in m o s t o f t h e f o l l o w i n g figures. D a t a o n t h e a c t i v a t i o n state o f R u b i s c o is p r e s e n t e d l a t e r (this p r o v i d e s i n f o r m a t i o n a b o u t t h e s y s t e m r e s p o n s e to a d e c r e a s e d a m o u n t o f R u b i s c o , b u t is n o t n e e d e d o r s u i t a b l e f o r c a l c u l a t i o n o f CRubi.J. A F i g u r e 1 s h o w s the r e l a t i o n b e t w e e n R u b i s c o c o n t e n t a n d C O 2 a s s i m i l a t i o n r a t e (A) at g r o w t h i r r a d i a n c e . P l a n t s g r o w i n g at h i g h e r i r r a d i a n c e c o n t a i n e d m o r e R u b isco, a n d p h o t o s y n t h e s i s e d at a h i g h e r rate. I f j u s t w i l d t y p e i n d i v i d u a l s a r e c o n s i d e r e d (solid s y m b o l s ) , w e see a near linear relation between A and Rubisco content. This w o u l d o f t e n be i n t e r p r e t e d as e v i d e n c e t h a t R u b i s c o is
M. L a u e r e r et al. : D e c r e a s e d R u b i s c o in t r a n s f o r m e d tobacco. VI I
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Quick et al. 1992) for CR,blsr A o f 0.03, 0.04 and 0.07 for plants g r o w n a n d m e a s u r e d at an irradiance o f 100, 300 and 750 pmol 9 m - z . s-1, respectively. We next investigated the effect o f short-term changes o f the irradiance. Light-response curves were m e a s u r e d in wild type plants a n d in 'antisense' t r a n s f o r m a n t s which had g r o w n at irradiances o f 100 (Fig. 2A), or 300 (Fig. 2B) or 750 (Fig. 2C) ~mol 9 m 2 . S 1. AS previously seen ( B j 6 r k m a n 1981 ; E v a n s 1989), the m a x i m u m rate o f photosynthesis was lower, and photosynthesis became saturated at lower irradiances in low-light-grown wild type plants than in high-light g r o w n wild types ( c o m p a r e Fig. 2 A with Fig. 2B and 2C). It m i g h t be noted that wild type individuals were light-limited when g r o w i n g at 100 and 300 lamol 9 m - 2. s - 1, b u t were close to light saturation when growing at 750 lamol, m - 2 . s -1. A t each g r o w t h irradiance, 'antisense' plants with decreased expression o f R u b i s c o b e c a m e light saturated at a lower irradiance, and h a d a lower m a x i m u m light-saturated rate o f photosynthesis. There was no large alteration in the initial slope o f the light response in 'antisense' plants g r o w i n g at 100 or 300 lamol 9 m -2 9 s 1. T h e initial slope was decreased slightly c o m p a r e d to the wild type in 'antisense' plants with very low R u b i s c o g r o w i n g at 750 g m o l 9 m -2 " S -1 In the experiment o f Fig. 3 the rate o f photosynthesis was m e a s u r e d at three different sho:rt-term irradiances (see legend) for a larger n u m b e r o f plants f r o m each growth-irradiance regime. In general, the flux control coefficient o f Rubisco increased (at !a given g r o w t h irradiance regime) when the m e a s u r i n g irradiance was increased, a n d decreased (at a given measuring irradiance) when the g r o w t h irradiance was increased. A t t e n t i o n is d r a w n to three further aspects o f the results. Firstly, when plants which had g r o w n at 300 or 750 ~tmol 9 m -2 9 s - a were exposed to 750 lamol 9m -2 " S - t a curvilinear relation between A and R u b i s c o was found, with only a small slope in the region c o r r e s p o n d i n g to the wild type (Fig. 3B, C). This can be contrasted with the A versus R u b i s c o plot for plants g r o w n at 100 and illumi-
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"limiting" for photosynthesis. H o w e v e r the additional i n f o r m a t i o n provided by the 'antisense' plants (open symbols) demonstrates that, in all three g r o w t h conditions, a considerable p o r t i o n o f the R u b i s c o can be r e m o v e d w i t h o u t leading tO a strong inhibition o f photosynthesis. Therefore, R u b i s c o c a n n o t limit the rate o f a m b i e n t photosynthesis at any o f these g r o w t h irradiances. The slope o f the A versus R u b i s c o plot in the wild type range gave an estimate (see Stitt et al. 1991;
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Fig. 2 A - C . I r r a d i a n c e - r e s p o n s e curves. T o b a c c o p l a n t s were g r o w n at 100 (A), 300 (B) or 750 (C) l a m o l - m Z . s 1 irradiance a n d illuminated at a r a n g e o f i r r a d i a n c e s , starting at the lowest. F o r each g r o w t h regime, the light r e s p o n s e s o f source leaves f r o m three different plants were m e a s u r e d , i n c l u d i n g o n e wild type (A, e , I ) a n d two ' a n t i s e n s e ' t r a n s f o r m a n t s with decreasing a m o u n t s o f R u b i s c o (A, 9 n). M a x i m a l R u b i s c o activity ( g m o l . m - z . s -1) was 29.6, 12.1 a n d 6.6 (at 100 g m o l - m 2. s 1 g r o w t h irradiance),
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336
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at 750 gmol. m z. s 1 and illuminated at 300 (o), 750 (m) or 1300 (o) lamol, m -2 - s-1. Plants were grown and photosynthesis was measured at 20~C. Each data point represents a separate plant. Solid symbols represent measurement at the growth irradiance
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Fig. 4A-C. Influence of Rubisco content on photosynthesis of t o b a c c o a t 28 ~ C . A P l a n t s w e r e g r o w n a t 100 g m o l 9 m - 2 - s - 1 a n d p h o t o s y n t h e s i s w a s m e a s u r e d a t 50 (V), 100 ( A ) o r 3 0 0 ( 9 l a m o l - m - 2 . s - 1. B P l a n t s w e r e g r o w n a t 3 0 0 g m o l . m - z . s i a n d p h o t o s y n t h e s i s w a s m e a s u r e d a t 100 ( A ) , 3 0 0 ( O ) o r 1000 ( O )
gmol - m- 2. s- 1. C Plants were grown at 1000 lamol, m - 2. s- 1 and photosynthesis was measured at 300 (0), 1000 (m) or 2000 (0) gmol 9m - 2. s - 1. Plants were grown and photosynthesis was measured at 28~ C. The ambient growth irradiance is always shown as a solid symbol. Each datum point represents a separate plant
n a t e d at 750 lamol 9m 2 . S - 1 (Fig. 3A). T h e y showed a linear r e l a t i o n b e t w e e n R u b i s c o activity a n d the rate of p h o t o s y n t h e s i s , which extended into the range corres p o n d i n g to the wild type (i.e. C RA u b i s c o --+ 1). T h u s , p l a n t s g r o w n in low light are o b v i o u s l y a l m o s t totally limited b y R u b i s c o w h e n s u d d e n l y exposed to high light. I n c o n t r a s t p l a n t s g r o w n at higher light have a d a p t e d to these c o n d i t i o n s ; they c o n t a i n m o r e R u b i s c o (see below for f u r t h e r charges) a n d are able to p h o t o s y n t h e s i s e in high i r r a d i a n c e w i t h o u t b e c o m i n g subject to a one-sided l i m i t a t i o n by R u b i s c o . Secondly, p h o t o s y n t h e s i s b e c a m e l i g h t - s a t u r a t e d at a b o u t 800 lamol" m - 2 " s - 1 w h e n p l a n t s were g r o w n at 300 ~tmol - m - 2 . S 1 , a n d at a b o u t 900 p.mol - m - 2 . s-1 w h e n wild type p l a n t s were g r o w n at 750 g m o l - m - Z . s 1 (Fig. 2). T h e rather low flux c o n t r o l coefficient o f R u b i s c o in the highest m e a s u r i n g i r r a d i a n c e in Figs. 3B a n d 3C therefore c o r r e s p o n d s to p l a n t s which are near, a n d completely, l i g h t - s a t u r a t e d (see below for m o r e data). Thirdly, w h e n p l a n t s which h a d g r o w n at 300 or 750 g m o l - m -2 9 s 1 were illumin a t e d at 100 g m o l 9m -2 9 s 1, the rate of p h o t o s y n t h e s i s was slightly higher in ' a n t i s e n s e ' t r a n s f o r m a n t s with decreased R u b i s c o c o n t e n t t h a n in the wild types. This effect was even larger w h e n p h o t o s y n t h e s i s was expressed
o n a structural dry-weight basis (data n o t shown, see also below, Fig. 6B, 6C). Growth and photosynthesis at 28 ~ C. In a separate experim e n t , three sets o f plants were g r o w n at 100, 300 a n d 1000 ~tmol 9 m - 2 . S - 1 irradiance at a higher t e m p e r a t u r e t h a n in the previous experiment, n a m e l y 28 ~ C. T h e rate o f p h o t o s y n t h e s i s was m e a s u r e d at 28 ~ C u n d e r growth i r r a d i a n c e (Fig. 4, closed symbols), a n d at lower a n d higher irradiance (open symbols). The following features were confirmed at this higher growth t e m p e r a t u r e . Firstly, Rubisco did n o t limit the rate of photosynthesis at g r o w t h irradiance. Values o f CRA,biscoo f 0.04, 0.14 a n d 0.24 c a n be estimated for p l a n t s g r o w i n g at 100 (Fig. 4A), 300 (Fig. 4B) a n d 1000 (Fig. 4C) ~ m o l - m - 2 " s -1, respectively. Secondly, R u b i s c o exerted little or n o c o n t r o l at low m e a s u r i n g light, a n d exerted a n increasing degree o f c o n t r o l w h e n the m e a s u r i n g light intensity was increased. Thirdly, w h e n p h o t o s y n t h e s i s is m e a s u r e d at a c o m m o n irradiance, plants g r o w n at low i r r a d i a n c e have a higher flux c o n t r o l coefficient for R u b i s c o t h a n plants g r o w n at high irradiance, (e.g. c o m p a r e the results for 300 ~ t m o l . m - z . s -1 in Figs. 4A, 4B a n d 4C). F o u r t h l y , high-light-grown leaves still have a rela-
M. Lauerer et al.: Decreased Rubisco in transformed tobacco. VI
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337
Chlorophyll fluorescence and N A D P - M D H activation.
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We next m e a s u r e d parameters which should provide i n f o r m a t i o n on the balance between CO2 fixation and electron transport. C h l o r o p h y l l fluorescence was measured in the experiment o f Fig. 3 at 20 ~ C, and resolved into p h o t o c h e m i c a l quenching (qQ) and the fast-relaxing c o m p o n e n t o f the n o n - p h o t o c h e m i c a l q u e n c h (qE) (Schreiber et al. 1986; Quick a n d Stitt 1990). Decreased R u b i s c o was a c c o m p a n i e d by an increase o f qE (Fig. 7 A - C ) , indicating that thylakoid energisation a n d associated p H - d e p e n d e n t energy dissipation is increasing. This change was already a p p a r e n t in plants with only a small reduction o f Rubisco. W h e n R u b i s c o was decreased further, q Q began to decrease too (Fig. 8A C), revealing that the acceptor side o f P S I I is b e c o m i n g m o r e reduced. The reduction state o f the acceptor side o f PSI was estimated by measuring the activation state o f N A D P - M D H , a thioredoxin-activated e n z y m e whose activation is inhibited by N A D P a n d hence provides a metabolic indicator for the stromal N A D P H / N A D P ratio (Scheibe and Stilt 1988). The activation state o f N A D P - M D H did n o t show a consistant increase until Rubisco c o n t e n t was strongly decreased (Fig. 9 A - C ) . Increasing the m e a s u r i n g irradiance also led to an increase in qE (Fig. 8), a decrease o f qQ (revealing increased reduction o f PSII) a n d increased activation o f N A D P - M D H (revealing increased reduction o f stromal N A D P ) . These changes were especially large in the wild type. W h e n plants g r o w n at different irradiances were c o m pared at a c o m m o n measuring irradiance, plants g r o w n at high light had lower qE (decreased thylakoid energisation), higher q Q (decreased reduction o f PSII) a n d lower NADP-MDH activation (decreased reduction o f the stromal N A D P ) than low-light-grown plants. These differences provide evidence that the leaves have altered the balance between light harvesting a n d energy utilisation in response to the g r o w t h irradiance.
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tively low value for CR,bis~o A even when they are lightsaturated; for example, when leaves f r o m plants g r o w n at 1000 pmol . m - z . s -1 were illuminated at 2000 ~tmol 9 m - 2. s - 1 the rate o f photosynthesis was n o t further increased, but the leaves h a d a CRAubiscoOfonly 0.12 (Fig. 4C). Specific leaf weight decreases in plants with less R u b isco (Fig. 5, see also Quick et al. 1991b). Therefore, when photosynthesis is expressed o n a leaf dry-weight basis, 'antisense' plants g r o w n at high irradiance have a slightly higher rate o f photosynthesis than the wild type, when photosynthesis is m e a s u r e d at 100 ~ t m o l . m -2 .s -1 (Fig. 6, see Fichtner et al. 1993 for a discussion o f the reasons for the c h a n g e o f specific leaf weight, a n d below for further implications for interpretation o f data). 1.5 Growth irradiance (I)
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