PhotosynthesisResearch23:205-212, 1990. © 1990KluwerAcademicPublishers.Printedin the Netherlands. Regular paper

Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves I. M A D H U S U D A N A

R A O , A. R A V I R A J A R U L A N A N T H A M

& NORMAN

TERRY 1

Department of Plant and Soil Biology, University of California, Berkeley, CA 94720, USA; I To whom offprint requests should be addressed Received 13 January 1989; acceptedin revised form 11 May 1989

Key words." adenylates, diurnal changes, nicotinamide nucleotides, photosynthetic induction, sugar beet Abstract

Sugar beets (Beta vuIgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25 °C, with a photon flux density of 500 #mol m 2S 1. Measurements were made of net CO2 exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD +, NADP +, NADH, NADPH), over the diurnal period (16 h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO2 uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 rain and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP + ; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP + and NADPH due to the light-driven phosphorylation of NAD + to NADP +. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP + (but not NAD + and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large cbange in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP + for intact leaves are low and constant during steady-state illumination.

Abbreviations; AEC-adenylate energy charge, DHAP-dihydroxyacetone phosphate, MTT-3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, PES - phenazine ethosulfate, PEP - phosphoenolpyruvate, PGA - 3-phosphoglycerate, PFD - photon flux density, Ru5P - ribulose-5-phosphate, Rubisco - ribulose 1,5bisphosphate carboxylase/oxygenase

Introduction

Adenylate and nicotinamide nucleotide pools inside the chloroplasts couple the light reactions of photosynthesis to the activity of the Calvin cycle (dark reactions). ATP and NADPH produced by the processes of photosynthetic electron transport are used in the reduction of CO2 to triose phosphate. In CO2-saturated photosynthesis each turn

of the Calvin cycle consumes 3 mol of ATP, 2 of which are used by PGA kinase and 1 by Ru5P kinase; in addition, 2 mol of NADPH are required per tool CO2 fixed for the reduction of PGA. During steady-state photosynthesis the rate of CO2 fixation and the rate of electron transport and photophosphorylation are matched as determined by the reaction stoichiometries of the Calvin cycle. One might expect that, under constant environ-

206 mental conditions, the rate of photosynthesis and the ATP/ADP and NADPH/NADP + ratios would remain constant throughout the light period. However, since many metabolic processes compete for the incoming supply of ATP and NADPH, and since the requirements of these metabolic pathways for ATP and NADPH could change during the day (e.g., change in triose-P utilization, (Leegood et al. 1985, Sharkey 1985)), it is conceivable that ATP/ ADP and NADPH/NADP ÷ ratios might also change diurnally. There are few published data showing how leaf adenylates and nicotinamide nucleotides change with time diurnally. Data for spinach indicate that ATP levels increase on illumination but subsequently vary with time (Bonzon et al. 1981) while in Lemna, ATP and total adenylates were higher in the light than in the dark but exhibited decreases with time during illumination (Kondo and Nakashima 1979). The objective of the present work therefore was to determine the diurnal changes in the pools of adenylates, and oxidized and reduced nicotinamide nucleotides, and to relate those changes to variation in the net rate of CO2 exchange.

Materials and methods

Plant culture and growth conditions Sugar beets (Beta vulgar& L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25 °C, 500 Ftmolm 2s 1 photon flux density (400700nm) and a 16h photoperiod (Terry 1980). Plants were cultured for two weeks after sowing in sand with distilled water. They were then transplanted (24 plants per 151 container) into a culture solution containing (millimol/liter): 2.5 Ca(NO3)2, 1.0 KH2PO4, 3.0 KNO3, 1.0 MgSO4, and 0.5 NaC1, and (micromol/liter): 23.1 H2BO3, 4.6 MnC12, 0.38 ZnSO4, 0.16 CuSO4, 0.052 H2MoO4, and 44.8 FeSO4 (as ferric-sodium EDTA complex). After 10 days, the plants were transferred (2 plants/201 container) to solutions with the same composition as the preceding one. The pH of the nutrient solution was maintained at about 6.0 by addition of solid CaCO3. Plants were grown for 10 days and the measurements were carried out using recently expanded leaves.

Leaf gas exchange The rate of photosynthetic CO2 uptake per unit leaf area (P/area) was determined at 500#molm 2s PFD, 30Pa CO2, 21KPa (21% 02) and 25°C as described previously (Terry 1983). Respiratory COz release was determined during darkness at 30Pa CO2, 21 KPa 02 and 25°C.

Leaf sampling and extraction of adenylate and nicotinamide nucleotides For extraction of adenylate and nicotinamide nucleotides in intact leaf tissue, samples were prepared after 8 h of continuous darkness at 25 °C in the growth chamber. The plants were then illuminated in the growth chamber at 500 #mol m 2s- 1 PFD and samples were prepared at 1, 5, 30min, and 1, 4, 8, 12, 16 h of illumination, and 2, 4, and 8 h of darkness. Samples were prepared as follows: at each time point 4 leaf discs (3.88 cmz each) were rapidly punched and frozen in liquid N2 using a light transmitting leaf disc punch machine (the entire process taking 1-2 s at growth chamber light/ dark conditions to ensure same light intensity and temperature during the killing procedure). For the extraction of adenylates and oxidized nicotinamide nucleotides (NADP+; NAD+), extracts were prepared by grinding the leaf material in 12% HC104 (4 discs per 4cm 3) in a liquid N2-cooled mortar and pestle. The extracts were left for 1 h on ice and centrifuged at 10,000g for 10min at 4°C. The supernatant was then neutralized with 10 mol/ liter KOH. The KC104 precipitate was removed from the extract by centrifugation in a microfuge. For the extraction of reduced nicotinamide nucleotides (NADPH; NADH), 1 mol/liter NaOH was used instead of HC104. The extracts were boiled for 5 min and rapidly cooled before centrifugation. The supernatants were neutralized with 6 N HC1.

Assay of adenylates Adenylate (ATP; ADP; AMP) levels were determined as described in Fader and Koller (1984) with some modifications. ATP was determined, after appropriate conversion reactions, for 100mm 3 ali-

207 quots using a sensitive, laboratory constructed photometer (courtesy of Dr. A. Melis) for the detection of the firefly luminescence from 500mm 3 reconstituted luciferin-luciferase (Sigma FLE-50) added to 2cm 3 reaction mixture (25mmol/liter Hepes-NaOH, pH 7.5; 25mmol/liter MgSO4; 10 mmol/liter K2 SO4; 5 mmol/liter EDTA). The instrument was routinely calibrated with internal standard ATP solutions. ADP was measured by enzymic conversion of ADP to ATP by pyruvate kinase, and then assayed for ATP. AMP was measured similarly by its stepwise conversion to ADP and then to ATP by myokinase and pyruvate kinase, respectively. Concentrations of the individual adenylates were determined by incubating 0.2 cm 3 of neutralized sample extracts in the following mixtures for 90 min at 25 °C prior to assay to ATP: (a) for ATP, 0.12 cm 3 of Hepes (0.2 mol/liter, pH 7.4), 0.02 cm 3 of 0.15 mol/liter MgClz, and 0.46 cm 3 of H20; (b) for ATP + ADP, 0.12cm 3 Hepes, 0.02 cm 3 MgC12, 0.01 cm 3 of PEP (10 mmol/liter), 0.0l cm 3 of pyruvate kinase (500 units in 0.5 cm 3 of l mmol/liter phosphate buffer, pH 7.4), and 0.44cm 3 H20; (c) for total adenylates (ATP + ADP + AMP), 0.12cm 3 Hepes, 0.02cm 3 MgC12, 0.01 c m 3 PEP, 0.01 cm 3 pyruvate kinase, 0.01 cm 3 myokinase (500units/cm3), and 0.43 cm 3 H20. The myokinase was desalted in a sephadex G-25 column before use. Sample ADP content was calculated by subtracting the original sample ATP content from the ATP content of the pyruvate kinase-converted sample. Sample AMP content was calculated from the difference of the ATP content of the pyruvate kinase-converted sample and the myokinase-pyruvate kinase-c0nverted sample. Adenylate energy change (AEC) was calculated according to Pradet and Raymond (1983): AEC = (ATP + 0.5ADP)/(ATP + ADP + AMP).

MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), 5 mmol/liter glucose-6phosphate, l mmol/liter phenazine ethosulfate (PES), and 200 mm 3 of sample in a final volume of 1.0 cm 3. The reaction was initiated by the addition of glucose-6-phosphate dehydrogenase (2.5 units). For the determination of NAD ÷ or NADH, the reaction medium was the same except that glucose6-phosphate was substituted by 96% ethanol (0.1 cm3). For the determination of NADH the medium contained 0.2 mM PES. The reaction was initiated by adding 66 or 33 units of alcohol dehydrogenase for the NAD ÷ or NADH assays, respectively. The rate of MTT reduction was recorded by measuring the absorbance change at 570 nm using an Aminco DW-2C spectrophotometer. The NADP + , NADPH, NAD ÷ , and NADH leaf tissue concentrations were calculated from corresponding standard curves of authentic substances.

Enzymes and chemicals All enzymes and reagents were products of Sigma Chemical Company.

Recovery of nucleotides The recovery of adenylates and nicotinamide nucleotides added to the leaf samples during extraction were determined. The recovery for adenylates was higher than 82% while for nicotinamide nucleotides was higher than 78%. The values reported were not corrected for losses during extraction and storage (at - 80 °C).

Results

Assay of nicotinamide nucleotides

Diurnal changes

The oxidized and reduced forms of the nicotinamide nucleotides were determined by enzymatic cycling as described by Maciejewska and Kacperska (1987) with slight modifications. The NADP + or NADPH contents were determined in a reaction medium containing: 30retool/liter Bicine-NaOH, pH 7.8, 0.5mmol/liter EDTA, 0.05mmol/liter

When the lights were switched on in the growth chamber, the rate of photosynthetic CO2 uptake, and the levels of ATP and NADPH, increased to a maximum in 30min and remained there for 16h without change (Fig. 1). When the lights were switched off, ATP and NADPH declined more slowly, requiring at least 4 h or longer to diminish

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LIGHT

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TIME IN HOURS Fig. 2. Diurnal changes sugarbeet plants. Values

LIGHT

A 0

4

to values approaching the lowest obtained in darkness (i.e., at 8 h). The increase in ATP on illumination was associated with a substantial increase in total adenylates (Fig. 2). If one compares the average light value with the average dark value (Table l), the 45% increase in ATP from light to dark was accompanied by a 41% increase in total adenylates; ADP increased 38% while AMP did not change significantly (Fig. 1B). The increase in the ratio of ATP/ADP on the other hand was relatively small; it increased by 5% from dark to light (Table 1). Similarly, the increase in NADPH on illumination (Fig. 1) was associated with an accumulation of

Y/ADARKZZ~~

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TIME IN HOURS Fig. 1. Diurnal changes in (A) leaf gas exchange/area (0); (B) leaf ATP (0), ADP (O), AMP (A); and (C)leaf NADPH (0) and NADP (0) of 5-week-old sugarbeet plants. Plants were illuminated at 500 pmol mm2 s-’ PFE. Values are mean + SD. from at least 3 replications. Leaf Chl/area was 427 mg m - 2 and dry wt/area was 3.6mgcm-‘.

Tub/e

I. Changes

in leaf adenylates

during

the diurnal

Treatment

ATP

ADP

Average Average

(pm01 m-*) 23.33 k 1.29 16.08 + 4.2

16.99 12.29

Table

mean

light dark

Changes & SD.

2.

Treatment

Average Average

light dark

in leaf nicotinamide

NADPH

NADP

(pm01 mm2) 2.24 + 0.17 1.49 & 0.24

3.28 2.47

nucleotides

+ 0.44 & 1.01

light and dark periods

& 1.69 F 0.75

during

the diurnal

NADH

NAD

2.23 &- 0.09 2.08 + 0.09

8.87 10.45

in leaf total adenylates of 5-week-old are mean F S.D. for 3 replications,

in 5-w-eek-old

AMP

Total

5.89 _+ 2.61 4.49 k 2.19

46.21 32.86

light

and dark

periods

Total

& 1.46 & 0.83

16.62 + 2.06 16.5 & 2.17

sugarbeet

plants.

k 2.32 k 2.77

in 5-week-old

Values

are mean

k S.D.

ATP/ADP

AEC

1.37 1.31

0.69 0.68

sugarbeet

plants.

NADPH NADP

NADH NAD

NADPH NADH

0.68 0.60

0.25 0.2

0.5 0.32

Values

+ NADP + NAD

are

209 both phosphorylated nicotinamide nucleotides (NADPH + NADP+); NADPH increased 50% from dark to light compared to a 33% increase in NADP ÷ (Fig. 1C). The NADPH/NADP-- ratio increased 13% on illumination; therfore, the increase in NADPH was only partly due to the reduction of NADP + (Table 2). Furthermore, the ratio of [NADPH + NADP+]/[NADH + NAD +] increased from 0.32 in the dark to 0.5 in the light while the total nicotinamides did not change between light and dark (Table 2).

ATP and NADPH would be due to the photophosphorylation of ADP to ATP and the photoreduction of NADP ÷ to NADPH rather than to biosynthesis of these nucleotides. We therefore investigated the kinetics of the changes of the nucleotides during the first 30 rain of illumination (Fig. 3; Table 3). The increase in photosynthetic rate and in ATP and NADPH levels when the lights are switched on, is very rapid, all three parameters increasing sharply in the first 60s (Fig. 3). Subsequently, photosynthetic rate increased at a slower rate over the next 29min. ATP and NADPH levels on the other hand stopped increasing or even decreased after the first 60 s, then increased again from 5 to 30 min (Fig. 3). All three parameters reached their maxima at 30 min. These high values were maintained without significant change for the remaining part of the 16h light period (Fig. 1B, C). Regression analysis indicated that the increase in leaf ATP

Photosynthetic induction The preceding data suggest that the build-up of adenylates and phosphorylated nicotinamide nucleotides occurred in less than 30min (Fig. 1). During the earliest stages of photosynthetic induction, one might expect that the accumulation of I

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TIME AFTER ILLUMINATION(MIN) Fig. 3. Changes in (A) photosynthesis/area (O); (B) l e a f A T P (o), A D P (El), A M P (zx); and (C) leaf N A D P H (o) and N A D P (El) during photosynthetic induction in 5-week-old sugarbeet leaves. Plants were dark adapted for 8 h before illumination at 500 #mol m - 2 s-~ PFD. Values are m e a n _+ S.D. for 4 replications.

210 Table 3. Changesin leaf adenylate and nicotinamide nucleotides during photosynthetic induction in 5-week-oldsugarbeetplants. Values are mean _+ S.D. for 4 replicates.

Nucleotides

Induction time (min) 0

1

5

30

Total

0zmolm 2) 30.91 _+ 3.8

34.9 - 2.0

32.1 + 1.3

44.41 _+ 2.0

ATP/ADP AEC

ratio 1.11 0.61

Adenylates

1.58 0.69

1.63 0.64

1.38 0.68

Nicotinamides NADH NAD Total NADPH/NADP NADH/NAD NADPH + NADP NADH + NAD

(#mol m -2) 2.01 + 0,5 9.87 4- 3.6 14.96 + 4.8 ratio 0.75 0.21 0.26

and total adenylates over the first 30 min after illumination was significant (P < 0.01). The 40% increase in A T P in the first 60 s was due to an increase in the A T P / A D P ratio which remained high up to 5 rain, then decreased to average light values at 30rain (Table 3). The increase in ATP from 5 to 30 rain appeared to be due to an increase in total adenylates and not to phosphorylation of A D P to ATP (Table 3). AEC values did not change with time (Table 3). The increase in N A D P H over the first rain was due to the reduction of N A D P +, the N A D P H / N A D P + ratio increasing by 55% (Table 3). The N A D P H / N A D P + ratio then declined from 1 to 5 min when it attained values a little lower than the average light value (Table 2). The increase in N A D P H from 5 to 30min was clearly attributable to the phosphorylation of N A D + to N A D P + as indicated by the significant increase in [ N A D P H + N A D P + ] / [ N A D H + N A D + ] ratio which occurred over that time (Table 3).

Discussion

The results suggest that illumination of intact leaves resulted in an increase in the total adenylate and [ N A D P H + N A D P +] (but not N A D + and N A D H ) pools. The accumulation of ATP in the

2.45 _+ 0.5 6.67 -t- 6.7 15.29 4- 8.4 1.16 0.25 0.26

2.12 + 0.2 8.87 + 4.8 15.13 4- 6.6 0.58 0.24 0.38

2.55 + 1.1 7.22 + 4.5 16,34 4- 7.4 0.62 0.35 0.67

light was not simply due to the phosphorylation of A D P to ATP but was in addition due to a net accumulation of adenylates. Conflicting data have been reported with respect to the influence of light on the steady-state A T P levels in photosynthetic tissues. Illumination increased the level of A T P (Walton et al. 1979, K o n d o and Nakashima 1979, Bonzon et al. 1981, Ulrich-Eberius et al. 1983), reduced the l e a f A T P (Van Bel et al. 1981), and had no effect (Luttge et al. 1971, Luttge and Ball 1976). The increase in total adenylates upon illumination for sugar beet leaves is consistent with the observations of Miginiac-Maslow and H o a r a u (1979) for wheat. The increase in A T P and N A D P H in the leaf on illumination almost certainly occurred as a result of changes within the chloroplast. Previous studies on determination of adenylate nucleotides in the chloroplast and nonchloroplast fractions of leaves or protoplasts indicated that an increase of the A T P / A D P ratio occurs only in the chloroplast (Santarious and Heber 1965, Stitt et al. 1982, H a m p p et al. 1982, 1984, 1985). H a m p p et al. (1982) showed that chloroplastic and cytosolic ATP increase rapidly on illumination while mitochondrial ATP decreases; light is thought to inhibit mitochondrial respiration (Gans and Rebeille 1988). In darkness, A T P levels in the chloroplast are maintained at reasonably high levels through a DHAP/PGA or oxaloacetate/malate shuttle

211 (Champigny 1978). The increase of N A D P ( H ) at the expense of N A D ( H ) on illumination is due to the phosphorylation of N A D + to N A D P + by a chloroplast-localized NAD-kinase which is lightactivated (Muto et al. 1981, Jarrett et al. 1982, Bonzon et al. 1983). The present study indicates that the increases in the pools of total adenylates and N A D P H / N A D P upon illumination was not accompanied by any large change in the energy or redox states of the system. It is generally believed that when plants are exposed to light, the ratios of A T P / A D P and N A D P H / N A D P + should increase and that this increase would be the driving force of photosynthesis. In fact the ratios do increase but not by very much as shown in the present work and by others (Lilley et al. 1977, Heber et al. 1982). Furthermore, photosynthesis may not be closely correlated with nucleotides: it has been shown that similar rates of CO2 fixation by intact chloroplasts (Kobayashi et al. 1979) and leaves (Kobayashi et al. 1982) can be observed at greatly different A T P / A D P ratios. The increase in leaf A T P or A T P / A D P ratio on illumination may be part of the mechanism whereby Rubisco becomes light activated (Brooks et al. 1988). The observation that AEC is not increased between light and dark is consistent w i t h the research of others who have shown that AEC is remarkably invariant under changing environmental conditions (Pradet and R a y m o n d 1983, R a y m o n d et al. 1987). Our results also indicate that the ratios of ATP/ A D P and N A D P H / N A D P + are low during the first five minutes of illumination in intact leaves (compared to the values obtained from isolated chloroplasts) and remain constant during steadystate photosynthesis. Values for the ratios of ATP/ A D P and N A D P H / N A D P + during induction were found to be higher for broken and intact chloroplasts compared to those of intact leaves (Pradet and R a y m o n d 1983, R a y m o n d et al. 1987). Transferring green leaves from the dark to the light produced very small changes in the AEC or in the corresponding A T P / A D P ratios of whole leaves (see Pradet and R a y m o n d 1983 for review). Our values are consistent with the whole leaf data. Heber et al. (1986) suggested that the reason for higher ratios for broken and intact chloroplasts is due to reduction in sinks for the consumption of ATP and N A D P H produced by the thylakoids.

Indeed Heber et al. (1986) found that when the rates of photosynthesis were high (e.g., in high light), the consumption of A T P and N A D P H resulted in low values of assimilatory force (which represents the phosphorylation potential and redox ratio). In the current conception of metabolic control (Kacser 1987), fluxes are thought to be of much greater significance than concentrations, ratios and equilibria. In other words, big changes in flux may be accompanied by apparently small changes in metabolic ratios. The regulation of flux depends not only on the kinetic behaviour of an enzyme towards the effectors, but also on the sensitivity of the activity of the enzyme to a change in flux.

Acknowledgements We thank C. Carlson for excellent technical assistance and Dr A. Melis and Ms B.M. Smith for advice on A T P measurements.

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