Planta
Planta (1986)167:414420
9 Springer-Verlag 1986
The influence of a decrease in irradiance on photosynthetic carbon assimilation in leaves of Spinacia oleracea L. R.T. Prinsley 1 *, S. Hunt 2, A.M. Smith 2 and R.C. Leegood 1 ** 1 Research Institute for Photosynthesis, Department of Botany, University of Sheffield, Sheffield, S10 2TN, and 2 John Innes Institute, Colney Lane, Norwich, NR4 4RU, U K
Abstract. When leaves of Spinacia oleracea L. were subjected to a decrease from a saturating to a limiting irradiance, photosynthetic carbon assimilation exhibited a pronounced lag. This comprised a postlower-illumination CO2 burst (Vines et al. 1982, Plant Physiol. 70, 629-631) and a slow increase in the rate of carbon assimilation to the new lower steady-state rate. The latter phenomenon was distinguishable from the former because it was present in leaves when photorespiration was inhibited by high concentrations of CO2 or by 2% 0 2 . A lag which followed a decrease in irradiance was also evident in leaves of Zea mays in air or in isolated spinach protoplasts photosynthesising in high COz. The lag was not stomatal in origin. The origin of the lag which followed the decrease in irradiance was investigated. Measurements of total 14CO2 fixation and 14C incorporated into sucrose during the transition in irradiance showed that sucrose synthesis displayed an overshoot during the transient which accounted for all of the carbon fixed during the first 90 s of the transition period. The behaviour of hexose phosphates in the intact leaf and in the cytosol was inconsistent with their supporting sucrose synthesis during the transient. It is concluded that the overshoot in sucrose synthesis imposes a drain on chloroplast intermediates which contributes to the temporary lag in the rate of carbon assimilation. Key words: Carbon dioxide assimilation - Light and carbon assimilation - Spinacia (photosynthesis) - Sucrose synthesis.
* Present address: Commonwealth Science Council, Marlbor-
ough House, Pall Mall, London SW1Y 5HX, U K ** To whom correspondence should be addressed A b b r e v i a t i o n : Ci = intercellular concentration of CO2
Introduction In the field, a leaf does not often experience a sudden transition between complete darkness and light, but the irradiance may fluctuate enormously. Causes for variations in irradiance in natural communities include changes in solar position, movement of clouds across the sun, and movement of leaves. The behaviour of photosynthetic processes in fluctuating light is not well understood. Most of the work done in this area has been prompted by interest in the effects of fluctuating light on whole-leaf carbon gain (McCree and Loomis 1969; Pollard 1970; Gross and Chabot 1979; Gross 1982). These workers found that certain levels and frequencies of light variations can contribute to daily carbon gain, but some also noted that, after transitions from a high to low irradiance, there was a lag in the rate of photosynthetic carbon assimilation of the kind first reported for Fucus by Steeman-Nielsen (1949). The lag is complicated by the presence o f a post-lower-illumination COz burst. Vines etal. (1982, 1983) have investigated the transitory release of CO2 from illuminated Geranium leaves when the irradiance was suddenly reduced. They found that the size of the CO2 burst was sensitive to changes in the concentrations of 02 and CO2, to irradiance and to temperature in a manner which suggested that the size of the COz burst is directly related to the rate of photorespiration. This "post-lower-illumination burst" is therefore analogous to the post-illumination burst of Decker (1955), seen when leaves are darkened. The extent to which the apparent lag in carbon assimilation is caused by post-lower-illumination COz burst is unclear. The most efficient response of carbon assimilation to a decrease in irradiance would be a simple step function, with carbon as-
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance similation immediately proceeding at the new l o w e r s t e a d y - s t a t e r a t e . S u c h a c h a n g e w o u l d require very rapid modulation of the amounts of photosynthetic intermediates and particularly of c y t o s o l i c i n t e r m e d i a t e s in t h e s y n t h e s i s o f s u c r o s e , because both the rate of sucrose synthesis and the a m o u n t s o f m e t a b o l i t e s in t h e c y t o s o l a r e m o r e sensitive to changes in light intensity than metabolites in t h e c h l o r o p l a s t ( S t i t t et al. 1983). I f a s t e p f u n c t i o n in t h e r a t e o f c a r b o n a s s i m i l a t i o n d o e s n o t o b t a i n , a n d t h e r e is a lag, t h e n t h i s w o u l d r e p r e s e n t a r e g u l a t o r y i m b a l a n c e in t h e s y s t e m a n d consequently a period of "lost" carbon assimilation. We have investigated the response of carbon a s s i m i l a t i o n t o a d e c r e a s e in i r r a d i a n c e in s p i n a c h l e a v e s a n d h a v e s h o w n t h a t it is n o t e n t i r e l y c a u s e d by a CO2 burst. We have therefore studied whether a n i m b a l a n c e b e t w e e n c a r b o n f i x a t i o n a n d its u t i l i s a t i o n in s u c r o s e s y n t h e s i s c o n t r i b u t e s t o t h e o b served lag by measuring the incorporation of 14C02 i n t o s u c r o s e a n d b y m e a s u r i n g t h e p o o l s of phosphorylated intermediates during the transient.
Materials and methods Plant material. Spinacia oleracea L. (cv. Virtuosa; Samuel Yates, Macclesfield, Cheshire, UK) was grown in hydroponic culture in a greenhouse with supplemental lighting with an l l - h light/13-h dark cycle. Large, mature leaves were harvested at the end of a light cycle and discs were cut from leaves under water with a sharp cork-borer. Other materials. Radiolabelled Bal~CO3 was obtained from Amersham International (Amersham, Bucks., UK). All other biochemicals were from Boehringer (Mannheim, FRG). Cellulase and macerozyme were obtained from Yakult Biochemicals (Nishinomiya, Japan). Bottled gases were from British Oxygen Company (BOC Special Gases, London, UK). Protoplasts and chloroplasts. Spinach protoplasts and chloroplasts were prepared and assayed as described by Leegood et al. (1981), except that the chloroplasts were further purified on a Percoll gradient as described by Mourioux and Douce (1981). Protoplasts (50 gg chlorophyll-ml-1) were assayed in a medium comprising 400mM sorbitol, 10 mM NaHCO3, 5mM CaC12, 50mM N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)-KOH (pH 7.6) at 20 ~ C. Chloroplasts (50 gg chlorophyll.m1-1) were assayed in 330mM sorbitol, 2 mM ethylenediarninetetraacetic acid (EDTA), 1 mM MgC12, 1 mM MnC12, 4 mM NaHCO3, 0.5 mM orthophosphate (Pi), 5 mM pyrophosphate (PPi), 2000 U catalase.m1-1, 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) (pH 7.6) at 20 ~ C. Gas-exchange measurements. The photosynthetic rate of spinach leaf discs (10 cm 2) at ambient levels of CO2 and 02 were measured with an ADC Mark III CO2 infra-red gas analyser (Analytical Development Co., Hoddeson, tterts., UK) in conjunction with an ADC Mark II water-vapour analyser. These were used in a chamber in an open system as described by
415 Harris et al. (1983). The intercellular concentration of CO2 (Ci) was estimated by the method of Cowan (1977). The apparatus used for pulse experiments with ~4CO2 on leaf discs with known, steady-state rates of photosynthesis was as described by Jones et al. (1983). Sixty 2.45-cm 2 leaf discs were cut from a large mature spinach leaf and darkened overnight. Two leaf discs were used for each sample. They were pre-illuminated, floating on distilled water, for 30 rain at the same irradiance as used during the gas-exchange measurements. The 14CO2 was generated from Bal~CO3 and was mixed with compressed air to fill a cylinder having a final CO2 concentration of 312 ~tl-1-1 at a specific activity of i MBq-1-1. Leaves were placed in the gas-exchange chamber, and when the rate of photosynthesis had been at steady-state for 3 rain, the leaf discs were pulsed with 14CO2 for 1 min at 1000 gmol quanta (400-700 nm)-m-Z.s 1, then the irradiance was decreased to 80 gmol-m-Z.s -1 while the pulse of 14CO2 was continued. At the end of the pulse of ~4CO2, the metabolism of the leaf discs was stopped with liquid Nz in the chamber (Jones et al. 1983). The temperature of the leaf was constantly monitored by a copper-constantan thermocouple touching the underside of the leaf. The rate of CO2 uptake in these experiments was monitored by an ADC Series 225 Mark 4 infra-red gas analyser. The leaf temperature was maintained at 20~ by a heat exchanger in the chamber walls which regulated the temperature of the air entering the chamber. The vapour-pressure deficit was maintained at 8 mbar.
Preparation of extracts. The frozen leaf material was ground to a fine powder in a pre-cooled mortar and pestle with a frozen pellet of 0.5 ml 1 M HCIO4 (Leegood and Furbank 1984). After the extract had thawed, the pestle and mortar were washed with three 150-gl aliquots of 0.1 M HC104, and the combined extract was centrifuged for 2 min at approx. 10000 g. The extracts were then neutralized (to pH 7) with 5 M K2CO3 and were centrifuged for 2 min at 2000 g. The 14C-labelled extracts were fractionated on ion-exchange resins by the method of Stitt and ap Rees (1978). Non-aqueous fractionation of leaves. Leaves were frozen in liquid N2, freeze-dried and fractionated using non-aqueous solvents as decribed by Dietz and Heber (1984). Metabolite assays. Metabolites were measured spectrophotometrically in a Hitachi 557 (Perkin-Elmer, Beaconsfield, Bucks., UK) dual-wavelength spectrophotometer (340400 nm) as described by Lowry and Passonneau (1972). Chlorophyll. Chlorophyll was measured by the method of Arnon (1949).
Results and discussion The characteristics o f carbon assimilation following a decrease in irradiance. W h e n t h e i r r a d i a n c e w a s decreased the time taken to reach a new steadys t a t e r a t e o f c a r b o n a s s i m i l a t i o n w a s f o u n d to b e greatest when the high irradiance saturated the rate of photosynthetic carbon assimilation and the lower irradiance strongly limited the rate of carbon a s s i m i l a t i o n . A d e c r e a s e f r o m 1 300 g m o l . m - 2. s - 1 t o 130 ~ m o l . m - 2. s - ~ r e s u l t e d i n a l a r g e i n d u c t i o n loss a n d a n e x t e n d e d l a g l a s t i n g 3 - 4 m i n ( F i g . 1 a).
416
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance
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Fig. 2. T r a c e a The effect of a decrease in irradiance from 1600 ~ m o l . m - 2 . s -1 to 80 g m o l - m - 2 " s -1 (red light) on the rate of CO2-dependent O2 evolution in isolated intact chloroplasts from spinach. T r a c e b The effect of a decrease in irradiance from 1600 g m o l . m - 2 - s -1 to 130 g m o l . m - 2 . s -1 on the rate of CO2-dependent O2 evolution by spinach protoplasts supplied with 10 m M NaHCO3. The a r r o w s indicate when the irradiance was lowered. N u m b e r s alongside the traces indicate rates of 02 evolution in p m o l - h - 1 - m g - I chlorophyll
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Fig. 1. a The effect of a decrease in irradiance from 1300 p.mol. m 2-s-1 to 130 lamol-m 2-s-1 on internal CO2 concentration (Ci) (---) and rate of CO2 uptake ( - - ) of a spinach leaf disc in air (320 pl. 1-1 CO2). The a r r o w indicates when the irradiance was lowered, b The effect of a decrease in irradiance from 1300 gmol. m - 2 "s 1 to 130 pmol- m - 2. s - 1 on leaf temperature (-.-) and transpiration ( . . . ) of a spinach leaf disc in air (320 gl1- t CO2)
Measurement of transpiration and leaf temperature indicated that physical factors were not responsible for the observed lag. The rate of transpiration decreased following a decrease in irradiance (Fig. l b) because stomata partially close in response to low irradiance. However, the lag cannot be explained by a decrease in stomatal conductance leading to a decreased intercellular concentration of COz (C~), since Ci actually increased sharply when the irradiance was lowered (Fig. 1 a). An increase in C~ following a decrease in irradiance may be due both to a decrease in the rate of COz assimilation and to processes generating CO2 internally, such as the post-lower-illumination CO2 burst. Since the calculation of Ci relies solely upon the measurement of stomatal conductance and the ex-
ternal CO2 concentration (Cowan 1977), any processes generating CO2 internally are likely to lead to an underestimation of C~. The estimates given in Fig. I a are therefore minimum values. Leaf temperature fell during the first minute following a decrease in irradiance. However, this fall was small (about 2 ~ in the experiment shown in Fig. 1 b) and it seems highly unlikely that it contributed to the lag in photosynthetic COg assimilation. These measurements indicate that the explanation for the lag lies in metabolic rather than physical constraints upon CO2 assimilation. The photorespiratory CO2 burst could not entirely account for the observed lag. Upon lowering the irradiance, there was an immediate decrease in the pool of glycine in leaves from 1200 to 500 n m o l . m g - 1 chlorophyll during the first 4 min at the lower irradiance (data not shown), which is consistent with a burst of photorespiratory COz production. However, a decrease in irradiance under conditions which inhibit photorespiration in leaves (2% O2 or 975 p l . l - t CO2 with 20% 02) and largely abolish the photorespiratory CO2 burst, still resulted in an appreciable lag (Figs. 3, 4). A lag following a decrease in irradiance either in 2% O2 or in 20% Oz was observed in the C4 plant Z e a mays, in which rates of photorespiration are extremely low (results not shown). Protoplasts
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance
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~
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Fig. 3. The effect of a decrease in irradiance from 1300 gmol. m - 2. s - 1 to 130 gmol. m - 2. s - 1 on the rate of COg assimilation by a spinach leaf disc in 2% 02 (320 Izl-1 1 CO2) or 20% 02 (320 gl. 1- z CO2). The a r r o w s indicate when the irradiance was lowered
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Fig. 4. Effect of a decrease in irradiance from 1300 ~tmol-m -2s - I to 430 ~tmol-m-g-s - I on the rate o f C O 2 assimilation by a spinach leaf disc in high COz (975 ~1.1-1 COz) in air. The l o w e r c u r v e shows photosynthesis at 430 ~tmol.m 2.s-1 including a dark-light induction period. The a r r o w indicates the time when irradiance was lowered
subjected to a decrease in irradiance under high COg usually showed a lag in photosynthetic COz assimilation (Fig. 2 trace b), again indicating that the photorespiratory COz burst was not the sole component of the lag and that metabolic rather than physical constraints are involved. However, no lag could be observed in COg-dependent O2 evolution in isolated chloroplasts following a decrease in irradiance (Fig. 2 trace a). These results indicate that, while the photorespiratory COz burst may play a part in the lag, other metabolic events
417
occurring outside the chloroplast are of major importance. Sucrose synthesis after a decrease in irradiance. Most of the triose phosphate generated by the chloroplasts during photosynthetic carbon assimilation must be retained in the chloroplast to regenerate ribulose-l,5-bisphosphate, but some may be transferred into the cytosol and converted to sucrose. The rate of sucrose synthesis is finely tuned to the rate of CO2 fixation, and therefore to the rate of triose-phosphate production (Stitt et al. 1983). If the rate of sucrose synthesis exceeds the rate of triose-phosphate production then depletion of stromal metabolites will occur, since free exchange of triose phosphate and orthophosphate is permitted by the phosphate translocator. If this occurs when the irradiance is decreased, the resulting drainage of triose phosphate from the Calvin cycle could impose the necessity for a period of metabolite build-up before steady-state photosynthesis is achieved. This possible explanation of the lag following a decrease in irradiance was tested by measuring changes in the rate of incorporation of 14CO2 into sucrose during the transient. The 14CO2 was supplied for I rain during steady-state carbon assimilation at a high irradiance and then the irradiance was decreased while the supply of 14COz continued. Supply of 14CO2 for a brief period prior to the decrease in irradiance allowed the subsequent small, immediate changes in the incorporation of ~4C into sucrose to be detected. Samples were taken immediately prior to the decrease in irradiance and over 7 min during the pulse of ~4COz at the low irradiance. The amount of 14C incorporated per I~mol CO2 assimilated during the 1-min pulse of ~4CO2 at high irradiance was calculated and the rate of CO2 assimilation at high irradiance was measured for all samples. These data were used to estimate the amount of ~4CO2 incorporated during the 1-min period of high irradiance for samples killed after the decrease in irradiance. This allowed the amount of ~4C incorporated in total and into sucrose at the low irradiance to be calculated. The pattern of incorporation of 14C into the leaf following the reduction in irradiance resembled the pattern of net CO2 fixation as measured by gas exchange (Fig. 5). However, incorporation of 14CO2 is likely to provide a slight underestimate of the net rate of CO2 assimilation immediately following the reduction in irradiance. This is because the pools of photorespiratory intermediates had not reached ~4C saturation at this point, so
418
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance
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Fig. 5. The a m o u n t o f c a r b o n entering into sucrose (o) a n d total c a r b o n fixation ( i ) as measured by 14C i n c o r p o r a t i o n following a decrease in irradiance. 14C was fed continuously to spinach leaf discs in air containing 312 gl. 1-1 CO2 for 1 min during steady-state photosynthesis at 1000 g m o l . m - 2. s - 1 a n d also after the irradiance was lowered to 80 g m o l - m - 2. s - 1. The time at which the irradiance was decreased is s h o w n as zero on the graph
Table 1. Total CO2 fixation and total c a r b o n fixed into sucrose (both estimated from the i n c o r p o r a t i o n o f 14CO2) measured during steady-state photosynthesis in low light (80 I x m o l - m - 2 . s - a ) and during the first 60 s following a decrease in irradiance from 1000 ~tmol-m - 2 . s -1 I n c o r p o r a t i o n o f 14C (nmol C- r a i n - t. m g - 1 chlorophyll) First 60 s Steady state at o f overshoot 80 gmol. m - 2. s - 1 Total CO2 fixation
375
518
C - a t o m s fixed into sucrose
456
238
that the ~4C supplied to the leaf would have been diluted internally with CO2 of a lower specific activity from the post-lower-illumination CO2 burst. Incorporation of 14C into sucrose following the reduction in irradiance continued for a brief period at the same rate as in high light before declining to a rate about half that in high light (Fig. 5 and results not shown, Table 1). The rate of incorporation of ~4C by the leaf was about equal to the rate of incorporation of ~4C into sucrose during the first few seconds after the reduction in irradiance, but considerably exceeded it after the lag had occurred. Calvin-cycle and cytosolic intermediates did not reach 14C saturation during the entire course of the experiment (results not shown). This means that movement of 14C into sucrose
provides a considerable underestimate of the actual rate of its synthesis relative to the rate of CO2 assimilation. The degree of underestimation is greatest at the point at which the degree of 14C saturation of the intermediates is least, that is, immediately following the reduction in irradiance. The fact that incorporation of 14C into sucrose at this point equalled the rate of 14C incorporation by the leaf means that the rate of sucrose synthesis must have been considerably in excess of the rate of CO2 assimilation. To determine whether this overshoot in sucrose synthesis immediately following a reduction in irradiance could lead to depletion of intermediates from the Calvin cycle, we investigated the source of carbon for sucrose synthesis during this period. Sucrose synthesis is supported by hexose phosphates present in the cytosol, which in turn derive from triose phosphate exported from the chloroplast in exchange for phosphate. Stitt et al. (1983) have shown that an overshoot in the rate of sucrose synthesis relative to CO2 assimilation following a decrease in CO2 concentration in wheat protoplasts may be due solely to consumption of cytosolic hexose phosphates. Hexose-phosphate synthesis from triose phosphate in the cytosol of these protoplasts stops immediately when the CO2 concentration is reduced because of very rapid inhibition of the cytosolic fructose-l,6-bisphosphatase. However, if the cytosolic fructose-l,6-bisphosphatase were not inhibited during an overshoot in sucrose synthesis, the overshoot would lead to a depletion of cytosolic, and hence chloroplastic, triose phosphate. We investigated the behaviour of hexose-phosphate and triose-phosphate pools in spinach leaves to discover whether cytosolic hexose phosphates alone could support the overshoot in sucrose synthesis following a decrease in irradiance or whether chloroplastic and other cytosolic intermediates were also involved. In the experiment shown in Fig. 5, the total amount of hexose phosphates (fructose-6-phosphate and glucose-6-phosphate) in leaves in high light was 170 n m o l ' m g -1 chlorophyll, but in low light, immediately following the lag in CO2 assimilation, the content was 132 n m o l . m g - t chlorophyll, a fall of only 38 n m o l . m g -1 chlorophyll (228 nmol C . m g - 1 chlorophyll). During this period the incorporation of carbon into sucrose, estimated from ~4C incorporation, was 456 nmol Cmg -1 chlorophyll (Table1) and, as discussed above, this was a considerable underestimate of the actual incorporation of carbon into sucrose. It is therefore clear that in this experiment the total decline in carbon in hexose phosphates following
R.T. Prinsley et al. : COz assimilation after a decrease in irradiance Table 2. The amount of hexose phosphate in the extra chloro-
plastic fraction of leaves extracted by non-aqueous means after irradiance was decreased from 1300 gmol.m- 2. s- 1 to 130 gmol'm-2-s -1 Time after a decrease in irradiance (s) 0 Hexose phosphates (nmol-mg-1 DW) 1.4 (nmol.mg l chlorophyll) 210
30
90
210
0.4 60
1.15 1.94 173 291
a decline in irradiance was very considerably less than the total incorporation of carbon into sucrose. To investigate the behaviour of pools of cytosolic intermediates, leaves which had undergone a reduction in irradiance were subjected to nonaqueous fractionation. Cytosolic hexose phosphates decreased by 150 nmol C" m g - 1 chlorophyll during the first 30 s after a decrease in irradiance, but then rose again so that after 210 s the amount was higher than before the transition (Table 2). The amounts of cytosolic triose phosphate fell from 15 to 6 nmol C . m g - a chlorophyll following the reduction in irradiance. Amounts of fructose1,6-bisphosphate were so low that they could not be reliably estimated. Changes in amounts of cytosolic intermediates following a reduction in irradiance could not therefore account for the overshoot in sucrose synthesis during this period. Since the net decrease in total amount of hexose phosphates, cytosolic hexose phosphates and total cytosolic intermediates were all considerably less than the increase in sucrose immediately following a decrease in irradiance, the overshoot in sucrose synthesis must be supported by drainage of intermediates from the chloroplast. Further evidence for this is provided by our finding that amounts of chloroplastic triose phosphate and hexose phosphates decreased substantially immediately following a reduction in irradiance (data not shown). Conclusions
The lag phase in net COa assimilation in spinach leaves following a reduction in irradiance cannot be accounted for by physical factors such as a photorespiratory CO2 burst, change in stomatal conductance or change in temperature, and must have a metabolic basis. Immediately following a reduction in irradiance, the rate of sucrose synthesis considerably exceeded the rate of CO2 assimilation. Decreases in the amounts of cytosolic intermedi-
419
ates of sucrose synthesis during this period were not sufficient to account for the overshoot in its synthesis, which must therefore involve considerable drainage of intermediates from the chloroplast. We conclude that depletion of pools of Calvin-cycle intermediates by an overshoot in sucrose synthesis, necessitating a period of metabolite build-up in the Calvin cycle before steady-state CO2 assimilation can be re-established at the lower irradiance, is one of the factors responsible for the lag phase following a reduction in irradiance. We would like to thank David Walker and Harold Woolhouse for their advice and criticism, Karl-Josef Dietz and Ulrich Heber for their help with the non-aqueous isolation of chloroplasts and Hitoshi Nakomoto for providing intact chloroplasts. This work was supported by the Agricultural and Food Research Council, UK, by the Science and Engineering Research Council, UK and by the Deutsche Forschungsgemeinschaft.
References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Cowan, I.R. (1977) Stomatal behaviour and environment. Adv. Bot. Res. 4, 117-228 Decker, J.P. (1955) A rapid, post-illumination deceleration of respiration in green leaves. Plant Physiol. 30, 82-84 Dietz, K-J., Heber, U. (1984) Rate-limiting factors in leaf photosynthesis. I. Carbon fluxes in the Calvin cycle. Biochim. Biophys. Acta. 767, 432-443 Gross, L.J., Chabot, B.F. (1979) Time course of photosynthetic response to changes in incident light energy. Plant Physiol. 63, 1033-1038 Gross, L.J. (1982) Photosynthetic dynamics in varying light environments: a model and its application to whole leaf carbon gain. Ecology 63, 84-93 Harris, G.C., Cheesbrough, J.K., Walker, D.A. (1983) Measurement of COz and H20 vapour exchange in spinach leaf discs. Effects of orthophosphate. Plant Physiol. 71, 102-107 Jones, D.A., Smith, A.M., Woolhouse, H.W. (1983) An apparatus for pulse and pulse-chase experiments with 14COz on attached leaves with known, steady-state rates of photosynthesis. Plant Cell Environ. 6, 161-166 Leegood, R.C., Edwards, G.E., Walker, D.A. (1981) Chloroplasts and protoplasts. In: Techniques in bioproductivity and photosynthesis, Coombs, J., Hall, D.O., eds. Pergamon Press, Oxford London Leegood, R.C., Furbank, R.T. (1984) Carbon metabolism and gas exchange in leaves of Zea mays L. Changes in CO2 fixation, chlorophyll a fluorescence, and metabolite levels during photosynthetic induction. Planta 162, 450-456 Lowry, O.H., Passonneau, J.V. (1972) A flexible system of enzymatic analysis. Academic Press, New York London McCree, K.J., Loomis, R.S. (1969) Photosynthesis in fluctuating light. Ecology 50, 422-428 Mourioux, G., Douce, R. (1981) Slow passive diffusion of orthophosphate between intact isolated chloroplasts and suspending medium. Plant Physiol. 67, 470-473 Pollard, D.F.W. (1970) The effect of rapidly changing light on the rate of photosynthesis in large tooth aspen (Populus grandidentata). Can. J. Bot. 48, 823-829 Steeman-Nielsen, E. (1949) A reversible inactivation of chlorophyll in vivo. Physiol. Plant. 2, 247-265
420 Stitt, M., ap Rees, T. (1978) Pathways of carbohydrate oxidation in leaves of P i s u m sativurn and Triticurn aestivurn. Phytochemistry 17, 1251-1256 Stitt, M., Wirtz, W., Heldt, H.W. (1983) Regulation of sucrose synthesis by cytoplasmic fructose bisphosphatase and sucrose phosphate synthase in varying light and carbon dioxide. Plant Physiol. 72, 767-774 Vines, H.M., Armitage, A.M., Chen, S., Tu, Z., Black, C.C. (1982) A transient burst of CO2 from Geranium leaves dur-
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance ing illumination at various light intensities as a measure of photorespiration. Plant Physiol. 70, 629-631 Vines, H.M., Tu, Z., Armitage, A.M., Chen, S., Black, C.C. (1983) Environmental responses of the post-lower illumination CO2 burst as related to leaf photorespiration. Plant Physiol. 73, 25-30 Received 8 October; accepted 5 November 1985
Planta (1986)167:414420
9 Springer-Verlag 1986
The influence of a decrease in irradiance on photosynthetic carbon assimilation in leaves of Spinacia oleracea L. R.T. Prinsley 1 *, S. Hunt 2, A.M. Smith 2 and R.C. Leegood 1 ** 1 Research Institute for Photosynthesis, Department of Botany, University of Sheffield, Sheffield, S10 2TN, and 2 John Innes Institute, Colney Lane, Norwich, NR4 4RU, U K
Abstract. When leaves of Spinacia oleracea L. were subjected to a decrease from a saturating to a limiting irradiance, photosynthetic carbon assimilation exhibited a pronounced lag. This comprised a postlower-illumination CO2 burst (Vines et al. 1982, Plant Physiol. 70, 629-631) and a slow increase in the rate of carbon assimilation to the new lower steady-state rate. The latter phenomenon was distinguishable from the former because it was present in leaves when photorespiration was inhibited by high concentrations of CO2 or by 2% 0 2 . A lag which followed a decrease in irradiance was also evident in leaves of Zea mays in air or in isolated spinach protoplasts photosynthesising in high COz. The lag was not stomatal in origin. The origin of the lag which followed the decrease in irradiance was investigated. Measurements of total 14CO2 fixation and 14C incorporated into sucrose during the transition in irradiance showed that sucrose synthesis displayed an overshoot during the transient which accounted for all of the carbon fixed during the first 90 s of the transition period. The behaviour of hexose phosphates in the intact leaf and in the cytosol was inconsistent with their supporting sucrose synthesis during the transient. It is concluded that the overshoot in sucrose synthesis imposes a drain on chloroplast intermediates which contributes to the temporary lag in the rate of carbon assimilation. Key words: Carbon dioxide assimilation - Light and carbon assimilation - Spinacia (photosynthesis) - Sucrose synthesis.
* Present address: Commonwealth Science Council, Marlbor-
ough House, Pall Mall, London SW1Y 5HX, U K ** To whom correspondence should be addressed A b b r e v i a t i o n : Ci = intercellular concentration of CO2
Introduction In the field, a leaf does not often experience a sudden transition between complete darkness and light, but the irradiance may fluctuate enormously. Causes for variations in irradiance in natural communities include changes in solar position, movement of clouds across the sun, and movement of leaves. The behaviour of photosynthetic processes in fluctuating light is not well understood. Most of the work done in this area has been prompted by interest in the effects of fluctuating light on whole-leaf carbon gain (McCree and Loomis 1969; Pollard 1970; Gross and Chabot 1979; Gross 1982). These workers found that certain levels and frequencies of light variations can contribute to daily carbon gain, but some also noted that, after transitions from a high to low irradiance, there was a lag in the rate of photosynthetic carbon assimilation of the kind first reported for Fucus by Steeman-Nielsen (1949). The lag is complicated by the presence o f a post-lower-illumination COz burst. Vines etal. (1982, 1983) have investigated the transitory release of CO2 from illuminated Geranium leaves when the irradiance was suddenly reduced. They found that the size of the CO2 burst was sensitive to changes in the concentrations of 02 and CO2, to irradiance and to temperature in a manner which suggested that the size of the COz burst is directly related to the rate of photorespiration. This "post-lower-illumination burst" is therefore analogous to the post-illumination burst of Decker (1955), seen when leaves are darkened. The extent to which the apparent lag in carbon assimilation is caused by post-lower-illumination COz burst is unclear. The most efficient response of carbon assimilation to a decrease in irradiance would be a simple step function, with carbon as-
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance similation immediately proceeding at the new l o w e r s t e a d y - s t a t e r a t e . S u c h a c h a n g e w o u l d require very rapid modulation of the amounts of photosynthetic intermediates and particularly of c y t o s o l i c i n t e r m e d i a t e s in t h e s y n t h e s i s o f s u c r o s e , because both the rate of sucrose synthesis and the a m o u n t s o f m e t a b o l i t e s in t h e c y t o s o l a r e m o r e sensitive to changes in light intensity than metabolites in t h e c h l o r o p l a s t ( S t i t t et al. 1983). I f a s t e p f u n c t i o n in t h e r a t e o f c a r b o n a s s i m i l a t i o n d o e s n o t o b t a i n , a n d t h e r e is a lag, t h e n t h i s w o u l d r e p r e s e n t a r e g u l a t o r y i m b a l a n c e in t h e s y s t e m a n d consequently a period of "lost" carbon assimilation. We have investigated the response of carbon a s s i m i l a t i o n t o a d e c r e a s e in i r r a d i a n c e in s p i n a c h l e a v e s a n d h a v e s h o w n t h a t it is n o t e n t i r e l y c a u s e d by a CO2 burst. We have therefore studied whether a n i m b a l a n c e b e t w e e n c a r b o n f i x a t i o n a n d its u t i l i s a t i o n in s u c r o s e s y n t h e s i s c o n t r i b u t e s t o t h e o b served lag by measuring the incorporation of 14C02 i n t o s u c r o s e a n d b y m e a s u r i n g t h e p o o l s of phosphorylated intermediates during the transient.
Materials and methods Plant material. Spinacia oleracea L. (cv. Virtuosa; Samuel Yates, Macclesfield, Cheshire, UK) was grown in hydroponic culture in a greenhouse with supplemental lighting with an l l - h light/13-h dark cycle. Large, mature leaves were harvested at the end of a light cycle and discs were cut from leaves under water with a sharp cork-borer. Other materials. Radiolabelled Bal~CO3 was obtained from Amersham International (Amersham, Bucks., UK). All other biochemicals were from Boehringer (Mannheim, FRG). Cellulase and macerozyme were obtained from Yakult Biochemicals (Nishinomiya, Japan). Bottled gases were from British Oxygen Company (BOC Special Gases, London, UK). Protoplasts and chloroplasts. Spinach protoplasts and chloroplasts were prepared and assayed as described by Leegood et al. (1981), except that the chloroplasts were further purified on a Percoll gradient as described by Mourioux and Douce (1981). Protoplasts (50 gg chlorophyll-ml-1) were assayed in a medium comprising 400mM sorbitol, 10 mM NaHCO3, 5mM CaC12, 50mM N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine (Tricine)-KOH (pH 7.6) at 20 ~ C. Chloroplasts (50 gg chlorophyll.m1-1) were assayed in 330mM sorbitol, 2 mM ethylenediarninetetraacetic acid (EDTA), 1 mM MgC12, 1 mM MnC12, 4 mM NaHCO3, 0.5 mM orthophosphate (Pi), 5 mM pyrophosphate (PPi), 2000 U catalase.m1-1, 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes) (pH 7.6) at 20 ~ C. Gas-exchange measurements. The photosynthetic rate of spinach leaf discs (10 cm 2) at ambient levels of CO2 and 02 were measured with an ADC Mark III CO2 infra-red gas analyser (Analytical Development Co., Hoddeson, tterts., UK) in conjunction with an ADC Mark II water-vapour analyser. These were used in a chamber in an open system as described by
415 Harris et al. (1983). The intercellular concentration of CO2 (Ci) was estimated by the method of Cowan (1977). The apparatus used for pulse experiments with ~4CO2 on leaf discs with known, steady-state rates of photosynthesis was as described by Jones et al. (1983). Sixty 2.45-cm 2 leaf discs were cut from a large mature spinach leaf and darkened overnight. Two leaf discs were used for each sample. They were pre-illuminated, floating on distilled water, for 30 rain at the same irradiance as used during the gas-exchange measurements. The 14CO2 was generated from Bal~CO3 and was mixed with compressed air to fill a cylinder having a final CO2 concentration of 312 ~tl-1-1 at a specific activity of i MBq-1-1. Leaves were placed in the gas-exchange chamber, and when the rate of photosynthesis had been at steady-state for 3 rain, the leaf discs were pulsed with 14CO2 for 1 min at 1000 gmol quanta (400-700 nm)-m-Z.s 1, then the irradiance was decreased to 80 gmol-m-Z.s -1 while the pulse of 14CO2 was continued. At the end of the pulse of ~4CO2, the metabolism of the leaf discs was stopped with liquid Nz in the chamber (Jones et al. 1983). The temperature of the leaf was constantly monitored by a copper-constantan thermocouple touching the underside of the leaf. The rate of CO2 uptake in these experiments was monitored by an ADC Series 225 Mark 4 infra-red gas analyser. The leaf temperature was maintained at 20~ by a heat exchanger in the chamber walls which regulated the temperature of the air entering the chamber. The vapour-pressure deficit was maintained at 8 mbar.
Preparation of extracts. The frozen leaf material was ground to a fine powder in a pre-cooled mortar and pestle with a frozen pellet of 0.5 ml 1 M HCIO4 (Leegood and Furbank 1984). After the extract had thawed, the pestle and mortar were washed with three 150-gl aliquots of 0.1 M HC104, and the combined extract was centrifuged for 2 min at approx. 10000 g. The extracts were then neutralized (to pH 7) with 5 M K2CO3 and were centrifuged for 2 min at 2000 g. The 14C-labelled extracts were fractionated on ion-exchange resins by the method of Stitt and ap Rees (1978). Non-aqueous fractionation of leaves. Leaves were frozen in liquid N2, freeze-dried and fractionated using non-aqueous solvents as decribed by Dietz and Heber (1984). Metabolite assays. Metabolites were measured spectrophotometrically in a Hitachi 557 (Perkin-Elmer, Beaconsfield, Bucks., UK) dual-wavelength spectrophotometer (340400 nm) as described by Lowry and Passonneau (1972). Chlorophyll. Chlorophyll was measured by the method of Arnon (1949).
Results and discussion The characteristics o f carbon assimilation following a decrease in irradiance. W h e n t h e i r r a d i a n c e w a s decreased the time taken to reach a new steadys t a t e r a t e o f c a r b o n a s s i m i l a t i o n w a s f o u n d to b e greatest when the high irradiance saturated the rate of photosynthetic carbon assimilation and the lower irradiance strongly limited the rate of carbon a s s i m i l a t i o n . A d e c r e a s e f r o m 1 300 g m o l . m - 2. s - 1 t o 130 ~ m o l . m - 2. s - ~ r e s u l t e d i n a l a r g e i n d u c t i o n loss a n d a n e x t e n d e d l a g l a s t i n g 3 - 4 m i n ( F i g . 1 a).
416
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance
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Fig. 2. T r a c e a The effect of a decrease in irradiance from 1600 ~ m o l . m - 2 . s -1 to 80 g m o l - m - 2 " s -1 (red light) on the rate of CO2-dependent O2 evolution in isolated intact chloroplasts from spinach. T r a c e b The effect of a decrease in irradiance from 1600 g m o l . m - 2 - s -1 to 130 g m o l . m - 2 . s -1 on the rate of CO2-dependent O2 evolution by spinach protoplasts supplied with 10 m M NaHCO3. The a r r o w s indicate when the irradiance was lowered. N u m b e r s alongside the traces indicate rates of 02 evolution in p m o l - h - 1 - m g - I chlorophyll
'5_
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~,~o
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Time (s)
Fig. 1. a The effect of a decrease in irradiance from 1300 p.mol. m 2-s-1 to 130 lamol-m 2-s-1 on internal CO2 concentration (Ci) (---) and rate of CO2 uptake ( - - ) of a spinach leaf disc in air (320 pl. 1-1 CO2). The a r r o w indicates when the irradiance was lowered, b The effect of a decrease in irradiance from 1300 gmol. m - 2 "s 1 to 130 pmol- m - 2. s - 1 on leaf temperature (-.-) and transpiration ( . . . ) of a spinach leaf disc in air (320 gl1- t CO2)
Measurement of transpiration and leaf temperature indicated that physical factors were not responsible for the observed lag. The rate of transpiration decreased following a decrease in irradiance (Fig. l b) because stomata partially close in response to low irradiance. However, the lag cannot be explained by a decrease in stomatal conductance leading to a decreased intercellular concentration of COz (C~), since Ci actually increased sharply when the irradiance was lowered (Fig. 1 a). An increase in C~ following a decrease in irradiance may be due both to a decrease in the rate of COz assimilation and to processes generating CO2 internally, such as the post-lower-illumination CO2 burst. Since the calculation of Ci relies solely upon the measurement of stomatal conductance and the ex-
ternal CO2 concentration (Cowan 1977), any processes generating CO2 internally are likely to lead to an underestimation of C~. The estimates given in Fig. I a are therefore minimum values. Leaf temperature fell during the first minute following a decrease in irradiance. However, this fall was small (about 2 ~ in the experiment shown in Fig. 1 b) and it seems highly unlikely that it contributed to the lag in photosynthetic COg assimilation. These measurements indicate that the explanation for the lag lies in metabolic rather than physical constraints upon CO2 assimilation. The photorespiratory CO2 burst could not entirely account for the observed lag. Upon lowering the irradiance, there was an immediate decrease in the pool of glycine in leaves from 1200 to 500 n m o l . m g - 1 chlorophyll during the first 4 min at the lower irradiance (data not shown), which is consistent with a burst of photorespiratory COz production. However, a decrease in irradiance under conditions which inhibit photorespiration in leaves (2% O2 or 975 p l . l - t CO2 with 20% 02) and largely abolish the photorespiratory CO2 burst, still resulted in an appreciable lag (Figs. 3, 4). A lag following a decrease in irradiance either in 2% O2 or in 20% Oz was observed in the C4 plant Z e a mays, in which rates of photorespiration are extremely low (results not shown). Protoplasts
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance
24
••'%
~
18
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oxygen
oxygen
I Time(mini
Fig. 3. The effect of a decrease in irradiance from 1300 gmol. m - 2. s - 1 to 130 gmol. m - 2. s - 1 on the rate of COg assimilation by a spinach leaf disc in 2% 02 (320 Izl-1 1 CO2) or 20% 02 (320 gl. 1- z CO2). The a r r o w s indicate when the irradiance was lowered
20
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Time{s)
Fig. 4. Effect of a decrease in irradiance from 1300 ~tmol-m -2s - I to 430 ~tmol-m-g-s - I on the rate o f C O 2 assimilation by a spinach leaf disc in high COz (975 ~1.1-1 COz) in air. The l o w e r c u r v e shows photosynthesis at 430 ~tmol.m 2.s-1 including a dark-light induction period. The a r r o w indicates the time when irradiance was lowered
subjected to a decrease in irradiance under high COg usually showed a lag in photosynthetic COz assimilation (Fig. 2 trace b), again indicating that the photorespiratory COz burst was not the sole component of the lag and that metabolic rather than physical constraints are involved. However, no lag could be observed in COg-dependent O2 evolution in isolated chloroplasts following a decrease in irradiance (Fig. 2 trace a). These results indicate that, while the photorespiratory COz burst may play a part in the lag, other metabolic events
417
occurring outside the chloroplast are of major importance. Sucrose synthesis after a decrease in irradiance. Most of the triose phosphate generated by the chloroplasts during photosynthetic carbon assimilation must be retained in the chloroplast to regenerate ribulose-l,5-bisphosphate, but some may be transferred into the cytosol and converted to sucrose. The rate of sucrose synthesis is finely tuned to the rate of CO2 fixation, and therefore to the rate of triose-phosphate production (Stitt et al. 1983). If the rate of sucrose synthesis exceeds the rate of triose-phosphate production then depletion of stromal metabolites will occur, since free exchange of triose phosphate and orthophosphate is permitted by the phosphate translocator. If this occurs when the irradiance is decreased, the resulting drainage of triose phosphate from the Calvin cycle could impose the necessity for a period of metabolite build-up before steady-state photosynthesis is achieved. This possible explanation of the lag following a decrease in irradiance was tested by measuring changes in the rate of incorporation of 14CO2 into sucrose during the transient. The 14CO2 was supplied for I rain during steady-state carbon assimilation at a high irradiance and then the irradiance was decreased while the supply of 14COz continued. Supply of 14CO2 for a brief period prior to the decrease in irradiance allowed the subsequent small, immediate changes in the incorporation of ~4C into sucrose to be detected. Samples were taken immediately prior to the decrease in irradiance and over 7 min during the pulse of ~4COz at the low irradiance. The amount of 14C incorporated per I~mol CO2 assimilated during the 1-min pulse of ~4CO2 at high irradiance was calculated and the rate of CO2 assimilation at high irradiance was measured for all samples. These data were used to estimate the amount of ~4CO2 incorporated during the 1-min period of high irradiance for samples killed after the decrease in irradiance. This allowed the amount of ~4C incorporated in total and into sucrose at the low irradiance to be calculated. The pattern of incorporation of 14C into the leaf following the reduction in irradiance resembled the pattern of net CO2 fixation as measured by gas exchange (Fig. 5). However, incorporation of 14CO2 is likely to provide a slight underestimate of the net rate of CO2 assimilation immediately following the reduction in irradiance. This is because the pools of photorespiratory intermediates had not reached ~4C saturation at this point, so
418
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance
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Fig. 5. The a m o u n t o f c a r b o n entering into sucrose (o) a n d total c a r b o n fixation ( i ) as measured by 14C i n c o r p o r a t i o n following a decrease in irradiance. 14C was fed continuously to spinach leaf discs in air containing 312 gl. 1-1 CO2 for 1 min during steady-state photosynthesis at 1000 g m o l . m - 2. s - 1 a n d also after the irradiance was lowered to 80 g m o l - m - 2. s - 1. The time at which the irradiance was decreased is s h o w n as zero on the graph
Table 1. Total CO2 fixation and total c a r b o n fixed into sucrose (both estimated from the i n c o r p o r a t i o n o f 14CO2) measured during steady-state photosynthesis in low light (80 I x m o l - m - 2 . s - a ) and during the first 60 s following a decrease in irradiance from 1000 ~tmol-m - 2 . s -1 I n c o r p o r a t i o n o f 14C (nmol C- r a i n - t. m g - 1 chlorophyll) First 60 s Steady state at o f overshoot 80 gmol. m - 2. s - 1 Total CO2 fixation
375
518
C - a t o m s fixed into sucrose
456
238
that the ~4C supplied to the leaf would have been diluted internally with CO2 of a lower specific activity from the post-lower-illumination CO2 burst. Incorporation of 14C into sucrose following the reduction in irradiance continued for a brief period at the same rate as in high light before declining to a rate about half that in high light (Fig. 5 and results not shown, Table 1). The rate of incorporation of ~4C by the leaf was about equal to the rate of incorporation of ~4C into sucrose during the first few seconds after the reduction in irradiance, but considerably exceeded it after the lag had occurred. Calvin-cycle and cytosolic intermediates did not reach 14C saturation during the entire course of the experiment (results not shown). This means that movement of 14C into sucrose
provides a considerable underestimate of the actual rate of its synthesis relative to the rate of CO2 assimilation. The degree of underestimation is greatest at the point at which the degree of 14C saturation of the intermediates is least, that is, immediately following the reduction in irradiance. The fact that incorporation of 14C into sucrose at this point equalled the rate of 14C incorporation by the leaf means that the rate of sucrose synthesis must have been considerably in excess of the rate of CO2 assimilation. To determine whether this overshoot in sucrose synthesis immediately following a reduction in irradiance could lead to depletion of intermediates from the Calvin cycle, we investigated the source of carbon for sucrose synthesis during this period. Sucrose synthesis is supported by hexose phosphates present in the cytosol, which in turn derive from triose phosphate exported from the chloroplast in exchange for phosphate. Stitt et al. (1983) have shown that an overshoot in the rate of sucrose synthesis relative to CO2 assimilation following a decrease in CO2 concentration in wheat protoplasts may be due solely to consumption of cytosolic hexose phosphates. Hexose-phosphate synthesis from triose phosphate in the cytosol of these protoplasts stops immediately when the CO2 concentration is reduced because of very rapid inhibition of the cytosolic fructose-l,6-bisphosphatase. However, if the cytosolic fructose-l,6-bisphosphatase were not inhibited during an overshoot in sucrose synthesis, the overshoot would lead to a depletion of cytosolic, and hence chloroplastic, triose phosphate. We investigated the behaviour of hexose-phosphate and triose-phosphate pools in spinach leaves to discover whether cytosolic hexose phosphates alone could support the overshoot in sucrose synthesis following a decrease in irradiance or whether chloroplastic and other cytosolic intermediates were also involved. In the experiment shown in Fig. 5, the total amount of hexose phosphates (fructose-6-phosphate and glucose-6-phosphate) in leaves in high light was 170 n m o l ' m g -1 chlorophyll, but in low light, immediately following the lag in CO2 assimilation, the content was 132 n m o l . m g - t chlorophyll, a fall of only 38 n m o l . m g -1 chlorophyll (228 nmol C . m g - 1 chlorophyll). During this period the incorporation of carbon into sucrose, estimated from ~4C incorporation, was 456 nmol Cmg -1 chlorophyll (Table1) and, as discussed above, this was a considerable underestimate of the actual incorporation of carbon into sucrose. It is therefore clear that in this experiment the total decline in carbon in hexose phosphates following
R.T. Prinsley et al. : COz assimilation after a decrease in irradiance Table 2. The amount of hexose phosphate in the extra chloro-
plastic fraction of leaves extracted by non-aqueous means after irradiance was decreased from 1300 gmol.m- 2. s- 1 to 130 gmol'm-2-s -1 Time after a decrease in irradiance (s) 0 Hexose phosphates (nmol-mg-1 DW) 1.4 (nmol.mg l chlorophyll) 210
30
90
210
0.4 60
1.15 1.94 173 291
a decline in irradiance was very considerably less than the total incorporation of carbon into sucrose. To investigate the behaviour of pools of cytosolic intermediates, leaves which had undergone a reduction in irradiance were subjected to nonaqueous fractionation. Cytosolic hexose phosphates decreased by 150 nmol C" m g - 1 chlorophyll during the first 30 s after a decrease in irradiance, but then rose again so that after 210 s the amount was higher than before the transition (Table 2). The amounts of cytosolic triose phosphate fell from 15 to 6 nmol C . m g - a chlorophyll following the reduction in irradiance. Amounts of fructose1,6-bisphosphate were so low that they could not be reliably estimated. Changes in amounts of cytosolic intermediates following a reduction in irradiance could not therefore account for the overshoot in sucrose synthesis during this period. Since the net decrease in total amount of hexose phosphates, cytosolic hexose phosphates and total cytosolic intermediates were all considerably less than the increase in sucrose immediately following a decrease in irradiance, the overshoot in sucrose synthesis must be supported by drainage of intermediates from the chloroplast. Further evidence for this is provided by our finding that amounts of chloroplastic triose phosphate and hexose phosphates decreased substantially immediately following a reduction in irradiance (data not shown). Conclusions
The lag phase in net COa assimilation in spinach leaves following a reduction in irradiance cannot be accounted for by physical factors such as a photorespiratory CO2 burst, change in stomatal conductance or change in temperature, and must have a metabolic basis. Immediately following a reduction in irradiance, the rate of sucrose synthesis considerably exceeded the rate of CO2 assimilation. Decreases in the amounts of cytosolic intermedi-
419
ates of sucrose synthesis during this period were not sufficient to account for the overshoot in its synthesis, which must therefore involve considerable drainage of intermediates from the chloroplast. We conclude that depletion of pools of Calvin-cycle intermediates by an overshoot in sucrose synthesis, necessitating a period of metabolite build-up in the Calvin cycle before steady-state CO2 assimilation can be re-established at the lower irradiance, is one of the factors responsible for the lag phase following a reduction in irradiance. We would like to thank David Walker and Harold Woolhouse for their advice and criticism, Karl-Josef Dietz and Ulrich Heber for their help with the non-aqueous isolation of chloroplasts and Hitoshi Nakomoto for providing intact chloroplasts. This work was supported by the Agricultural and Food Research Council, UK, by the Science and Engineering Research Council, UK and by the Deutsche Forschungsgemeinschaft.
References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 Cowan, I.R. (1977) Stomatal behaviour and environment. Adv. Bot. Res. 4, 117-228 Decker, J.P. (1955) A rapid, post-illumination deceleration of respiration in green leaves. Plant Physiol. 30, 82-84 Dietz, K-J., Heber, U. (1984) Rate-limiting factors in leaf photosynthesis. I. Carbon fluxes in the Calvin cycle. Biochim. Biophys. Acta. 767, 432-443 Gross, L.J., Chabot, B.F. (1979) Time course of photosynthetic response to changes in incident light energy. Plant Physiol. 63, 1033-1038 Gross, L.J. (1982) Photosynthetic dynamics in varying light environments: a model and its application to whole leaf carbon gain. Ecology 63, 84-93 Harris, G.C., Cheesbrough, J.K., Walker, D.A. (1983) Measurement of COz and H20 vapour exchange in spinach leaf discs. Effects of orthophosphate. Plant Physiol. 71, 102-107 Jones, D.A., Smith, A.M., Woolhouse, H.W. (1983) An apparatus for pulse and pulse-chase experiments with 14COz on attached leaves with known, steady-state rates of photosynthesis. Plant Cell Environ. 6, 161-166 Leegood, R.C., Edwards, G.E., Walker, D.A. (1981) Chloroplasts and protoplasts. In: Techniques in bioproductivity and photosynthesis, Coombs, J., Hall, D.O., eds. Pergamon Press, Oxford London Leegood, R.C., Furbank, R.T. (1984) Carbon metabolism and gas exchange in leaves of Zea mays L. Changes in CO2 fixation, chlorophyll a fluorescence, and metabolite levels during photosynthetic induction. Planta 162, 450-456 Lowry, O.H., Passonneau, J.V. (1972) A flexible system of enzymatic analysis. Academic Press, New York London McCree, K.J., Loomis, R.S. (1969) Photosynthesis in fluctuating light. Ecology 50, 422-428 Mourioux, G., Douce, R. (1981) Slow passive diffusion of orthophosphate between intact isolated chloroplasts and suspending medium. Plant Physiol. 67, 470-473 Pollard, D.F.W. (1970) The effect of rapidly changing light on the rate of photosynthesis in large tooth aspen (Populus grandidentata). Can. J. Bot. 48, 823-829 Steeman-Nielsen, E. (1949) A reversible inactivation of chlorophyll in vivo. Physiol. Plant. 2, 247-265
420 Stitt, M., ap Rees, T. (1978) Pathways of carbohydrate oxidation in leaves of P i s u m sativurn and Triticurn aestivurn. Phytochemistry 17, 1251-1256 Stitt, M., Wirtz, W., Heldt, H.W. (1983) Regulation of sucrose synthesis by cytoplasmic fructose bisphosphatase and sucrose phosphate synthase in varying light and carbon dioxide. Plant Physiol. 72, 767-774 Vines, H.M., Armitage, A.M., Chen, S., Tu, Z., Black, C.C. (1982) A transient burst of CO2 from Geranium leaves dur-
R.T. Prinsley et al. : CO2 assimilation after a decrease in irradiance ing illumination at various light intensities as a measure of photorespiration. Plant Physiol. 70, 629-631 Vines, H.M., Tu, Z., Armitage, A.M., Chen, S., Black, C.C. (1983) Environmental responses of the post-lower illumination CO2 burst as related to leaf photorespiration. Plant Physiol. 73, 25-30 Received 8 October; accepted 5 November 1985
