Photosynthesis Research 13:81-89 (1987) © Martinus Nijhoff Publishers, Dordrecht--Printed in the Netherlands

81

Regular paper

Effects of periodic fluctuations of photon flux density on anatomical and photosynthetic characteristics of soybean leaves J.P. G A U D I L L E R E , 1 J.J. D R E V O N , 2 J.P. B E R N O U D , 3 F. J A R D I N E T 3 & M EUVRARD 3 1Laboratoire de Chimie Biologique et de Photophysiologie, INRA, Centre de Grignon, F.78850 Thiverval, France, 2Laboratoire de Recherches sur les Symbiotes des Racines, INRA Place Viala, F.34060 Montpellier, France," SRhone Poulenc Agrochimie, 14 rue Pierre Baizet, F. 69009 Lyon, France

Received 9 December 1986; accepted in revised form 17 February 1987 Key words: Glycine max, fluctuating light, CO 2 conductance, Rubisco Abstract. The development of soybean leaves grown at fluctuating photon flux density between 100

and 1500#M m- 2s - ~with a period of 160 sec were compared to leaves developed under continuous light with the same mean photon flux density. Number of epidermal cells and stomata, leaf area and specific leaf weight were not affected by the periodic fluctuation of photon flux density. Chloroplastic pigment concentration and chlorophyll fluorescence reveal some photoinhibitory effects of the high photon flux density phase. Stomatal and internal CO 2conductance and the quantum yield were not affected by the light regime. In contrast ribulose 1.5 bisphosphate carboxylase/oxygenase activity before in vitro activation by CO 2 and Mg +÷ was stimulated by the periodic illumination whereas the total amount of the enzyme and the internal leaf CO 2conductance remained steady. In conclusion, there was no major difference between leaves of plant grown either under a steady or under a periodic fluctuation of the photon flux density except some photoinhibitory symptoms under fluctuating illumination, and a higher in vivo level of activation of the Rubisco.

Introduction

P l a n t g r o w n u n d e r n a t u r a l c o n d i t i o n s are subjected to a p e r m a n e n t fluctuation o f the p h o t o n flux density ( P F D ) . The light a d a p t a t i o n o f the p h o t o s y n t h e t i c a p p a r a t u s has been well d o c u m e n t e d u n d e r steady artificial a n d n a t u r a l illum i n a t i o n [1, 10]. H i g h light g r o w n s o y b e a n leaves are thicker, specific leaf weight, p r o t e i n c o n t e n t a n d R i b u l o s e 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 ( R u b i s c o ) activity are increased [28]. It is generally f o u n d that in response to high light, s t o m a t a l a n d internal CO2 c o n d u c t a n c e are enhanced. P h o t o s y s t e m s s t o i c h i o m e t r y is also affected by P F D [13]. The d i s t r i b u t i o n o f c h l o r o p l a s t v o l u m e between the m e m b r a n e a n d the s t r o m a phases is increased at high P F D . W h e n P F D change f r o m high to low a n d vice versa the p h o t o s y n t h e t i c characteristics a n d the leaf a n a t o m y a d a p t to some extent a n d reach a new equilibrium after some time [18]. N e t CO2 exchange by leaves u n d e r fluctuatin P F D has been studied [6, 9, 15, 17, 19, 27], b u t m a n y f u n d a m e n t a l aspects o f p h o t o s y n t h e t i c s characteristics u n d e r this i l l u m i n a t i o n are u n k n o w n . D o the leaves a d a p t to sun type or shade type u n d e r p e r m a n e n t fluctuations o f P F D ? The answer to this

82 question is needed to study growth of natural leaves and also to design efficient growth chambers for experimental or commercial objectives. In this paper we report some leaf characteristics of 40-day-old soybean plants grown under intermittant PFD generated by moving a lamp in a growth chamber in comparison to those of soybean plants grown under steady PFD.

Materials and methods

(a) Plant culture Inoculated plants (Glycine max (L.) Merr. cv Hodgson) were grown hydroponically in 1 liter pot. During the first 28 days of growth nitrogen was supplied as urea (1 mM) in the nutrient solution. After plants were totally deprived of nitrogen [12], the liquid root medium was continuously aerated by compressed air. The growth chamber, 3 m wide and 6 m long, was illuminated by a mobile beam carrying 13 metal halide lamps (1 kw HID General Electric). The beam travelled through the length of the chamber at a steady velocity of 4.5 m per min. This device is patented by Rhone Poulenc Agrochimie Company and is manufactured by Retma Company (Loire sur Rhone, 66700 Givors, France). In the center of the chamber the illumination varied between one minimum (100#M Photonsm-2s -~) and two maxima (1500#M photonsm-2s -1) at each cycle (Fig. la). The mean PFD measured on one cycle was 400#Mm-2s -~. The plants were grown at a photoperiod of 15 hours; the day and night temperature were respectively 25 °C and 20 °C; the relative humidity was around 70%. The circulation of the beam caused also fluctuation of the leaf temperature shown in Fig. lb, which is due to the very high PFD of the lamps. Steady illumination was obtained in a similar growth chamber with a stationary lamp beam, at 220, 400 and 800#M Photonsm-2s -~. 30

I~ I

I

il

~J IE1 -

I~

a I II

q t I I

~I

l

l[

I

'!

I

fl

,,

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i!

G

f;

20

2 w 10.

,

~\11

L

~,\1

\U

'

I

I

~d

I

0

i

0 TIME

(MIN)

TIME

4

(MIN)

I

B

Fig. 1. (a) Time course of PFD at the center of the growth chamber during the displacement of the beam. (b) Time course of the leaf temperature.

83

(b) Photosynthesis measurements Photosynthesis was measured on attached central leaflet of recently completely developed leaves. A CO2 concentration of 40 to 1100/d 1-~ in the assimilation chamber was obtained by mixing dry CO2 free air with an air enriched with 5% CO2. A vapor pressure of 1.7 kPa was obtained by adjusting the CO2 free air flow rate through the cuvette. The difference in vapor pressure between the leaf and the air was never higher than 0.6 kPa. The flow rates were controlled by mass flow meters Tylan (Logan, USA). Air dew point was measured by a General Eastern automatic dew point meter. CO2 was measured by an I R G A (Binos, Leybold Heraus Germany). Leaf temperature was measured by a 0.3 mm diameter thermocouple (Cu/Ct) pressed on the abaxial side of the leaf. Photosynthetic responses were recorded on the same leaf successively by increasing CO2 concentration and internal CO2 conductance were computed according Farquhar and Von Caemmerer [3, 4]. Leaf temperature was 20 °C.

(c) Pigments analysis and measurement of fluorescence Chla, Chlb and total carotenoids were measured by spectrophotometry on leaf samples extracted by N,N Dimethylformamide without grinding [11, 5]. Chlorophyll fluorescence in intact leaves was excited by red light produced by a laser He/Ne at a P F D of 200 #M m-2 s- ~. Photosynthetic electron transfer was blocked by infiltration of the leaves with atrazine (Norflurazon, Bayer). Fluorescence collected by an optical fiber was measured at wavelength above 700 nm. The yield of fluorescence is related to the density of intact photosystem II reaction centers [14].

(d) Measurements of activity and amount of Rub&co Rubisco activity was measured on leaf extracts after a 6 hours light period using the method of Perchorowicz et al. [16]. The activity was measured just after the leaves were crushed in the buffer. The lag between leaf sampling and Rubisco assay was never more than 5rain. The initial and the total activities of the enzyme incubated with CO2 and Mg ++ for 5 min were measured on the same extract. After the centrifugation, the leaf extract was stored at - 1 5 °C for further analysis of total amount of Rubisco. The protein of 550 kDa was separated as native form on polyacrylamide gel (4.5%) by electrophoresis according to the technic of Cammaert and Jacob [2]. The protein band was stained by Coomassie blue (0.2% Blue, 50% methanol, 10% acetic acid) for 1 hour. The gel was destained in a solution of 50% methanol and 10% acetic acid during 15 hours. The blue band was cut out and the blue color was solubilized in 3 ml of methanol 50%, at 50 °C for 2 hours. The optical density of the solution was measured

84 spectrophotometrically at 592 nm. The calibration of the amount of Rubisco was obtained with known amounts of Spinach Rubisco (Sigma) on the same gel (Fig. 2).

(e) Studies of the partitioning of photosynthetic carbon in the form of soluble sugars and starch Short term partitioning of carbon was studied by in situ incorporation of 14CO2 in leaves during 10 seconds. The radioactivity distribution was essayed 10min later. Sugars were solubilized in 50% methanol. Starch was digested by fl glucosidase (Merck) action [25]. Previously leaf residues were wetted and heated (10 rain, 95 °C) to make the digestion more efficient. The reaction lasts 24 hours at 37 °C at pH 4.6 in acetate buffer. The fl glycosidase amount was more than 70 U per mg of starch in the sample. The remaining insoluble radioactive fraction was solubilized by N,N Dimethylformamide. The extracted fraction was mainly composed of proteins and lipids. Radioactivity was measured by liquid scintillation in OCS scintillator (Amersham, UK) with a M R 300 counter (Kontron CH). The kinetic of export of recent assimilates was recorded by measuring the leaf radioactivity with a Geiger counter 24 hours after the labelling with T4CO2. The kinetic of the radioactivity disappearance was adjusted to a simple exponential law [20] and the turn over time of assimilates was computed.

(f) Determination of anatomical and water relationships parameters The size of epidermic cells on both sides of the leaves was measured on prints made by contact. Countings were done on 4 leaves and on 6 different locations on each leaves. Leaf water potential was measured with a Scholender bomb in the growth chamber [23, 29]. The osmotic potential was assayed by freezing temperature .15

o.05 o

HB

5 Rub~ sco

10

Fig. 2. Calibration curve of Rubisco quantification technic by the Coomassie blue eluted from protein spots on polyacrylamidegel.

85 Table 1. Environmental parameters and water status of 40 days old plants (5 replicates, + / confidence interval with a probability of 95%).

Irradiance Leaf Root Water potential (#M m z s t) temperature temperature (°C) (°C) Basal MPa

Mini MPa

Minimal turgor Mpa

Constant 220 400 800

25 25 29

25.5 27 28

-0.40 +/-0.40 +/-0.40 +/-

0.05 0.05 0.05

-0.60 +/-0.60 +/-0.75 +/-

0.18 0.55 + / 0.15 0.55 + / 0.20 0.22 + / -

0.20 0.20 0.25

Fluctuating 400

25-29

31.0

-0.35 +/-

0.05

-0.55 +/-

0.16 0.60 + / -

0.18

measurement with a microosmometer (Roebling gmbh, Germany). Thawed leaves were pressed in a 2-ml plastic syringe to extract the sap. The assay was performed on a 50/A distilled water solution containing 10#1 of sap. Turgescence was computed as the difference between water and osmotic potentials [29]. Results and discussion

Mean P F D had a direct effect on plant temperature and water potential (Table 1). The fluctuation introduced a continuous change in the leaf temperature with a amplitude of 5 °C maximum (Fig. lb), during the day time only. At the end of the night the water potential was not changed by either steady PFD of 220, 400 and 800 #M m - 2s- 1 or fluctuating irradiance of 400/tM m -2 s - J. At the end of the day turgor decreased with the increasing PFD level. The night recovery of the water potential showed that plants were not subjected to a permanent severe water stress, which is in agreement with the absence of PFD effects on the size of epidermic cells (Table 2). Cell enlargement was not affected by differences in turgor at the end of the day. With increasing PFD, the mean leaf area decreased, but the plant total leaf area and stomata number increased. This is a well known response to PFD and it was not affected by the PFD fluctuation: Table 2. Leaf anatomical characters of 40 days old Soybean grown at constant or fluctuating PFD (5 replicates, + / - confidence interval with a probability of 95%).

Irradiance Leaf area ~ M m-2 s-1 ) per plant (cm -2)

Mean leaf area (cm -2)

Number of epidermic cells (mm -2)

Constant 220 400 800

340 + / - 5 0 499 + / - 1 2 1 670 + / - 1 5 9

64.5 + / - 9 . 8 58.0 + / - 1 4 . 4 55.8 + / - 1 3 . 3

1390 + / - 1 8 9 1415 + / - 1 4 4 1282 + / - 1 1 5

Fluctuating 400

490 + / -

59.7 + / -

1390 +/-

70

8.5

Number of stomata (mm 2) Sup.

tnf

54 + / - 1 6 108 + / - 1 1 136 + / - 3 9

240 + / - 1 8 270 + / 27 286 + / - 5 5

178 119 + / -

37 231 + / -

45

86 in the experiment the plant respond typically to mean PFD for all these anatomic characteristics (Table 3). Stomatal and internal CO2 conductance increased with PFD. The sharp increase of the conductances between 400 and 800#Mm-2s -1 must be noticed. The photosynthetic apparatus adaptation capacities are not saturated by our experimental conditions. Fluctuation of PFD again did not affect the stomatal and internal CO2 conductance response of soybean to PFD. Rubisco activity was related to internal conductance in agreement with the classical biochemical models of photosynthesis [3]. This correlation is observed under permanent illumination (Table 2). The high PFD induced a 100% increase in the amount of enzyme. This increase could explain the concommitant increase of 100% in the total activity. On the other hand the percent of activation of the Rubisco decreased with the higher PFD. At high PFD Rubisco activity is not the only limiting factor since its activity of 156#M CO2 m 2S 1 at 800#Mm-2s -1 of photons is 5 times higher than the leaf photosynthetic activity permitted by the measured leaf conductance. Triose phosphates utilization is known to regulate the Rubisco activity [24]. Under fluctuating PFD the initial activity of Rubisco was sharply increased. The Rubisco amount of 1.35 gm -2 agrees with the PFD response of soybean leaves and is consistant with the total activity. Fluctuating light induced a discrepancy between the caroboxylase activity and the leaf internal CO2 conductance. Activation of Rubisco in soybean leaves is a complex phenomenon. The catalycally active complex involves CO2 and Mg ++. The binding of a specific inhibitor, 2-carboxy-D-arabinitol-1-phosphate has been recently discovered. This binding to the protein occurs during the night and is removed by light [21, 22, 30, 31, 10]. Our results could be interpreted as a direct effect of fluctuating illumination on the removing of this inhibitor. This hypothesis is under investigation. The photochemical apparatus also reacts with the PFD (Table 4). The chlorophyll amount and the ratio Clha/Chlb increased with PFD. This ratio reflects the increase with light of the proportion of PSII/PSI reaction center [13]. Such variation could be related to the simultaneous increase of the quantum yield. Under fluctuating light, soybean leaves appear to lose pigments. Their chlorophyll ratio is that of high light grown plants, but the chl/Carotenoids ratio Table 3. Stomatal and internal CO 2 conductance of soybean leaves (5 replicates), 40 days old. Rubisco activity is measured after 6 hours of light (3 replicates, + / - confidence interval with a probability of 95%). Irradiance (tzM m 2 s I)

Stomatal conductance (104ms I)

Internal conductance (104ms_l)

Rubisco activity

Initial Constant 220 400 800 Fluctuating 400

7,53 7.35 34.5 9.87

Leaf rubisco gm-2

(/IMCO2m 2 s 1)

% activ. Total

+ / - 0,15 + / - 0.28 + / - 0.06

3.25 9.43 34.5

+/ 2.8 + / 3.5 + / - 3.1

105 114 156

+ / 26 + / 71 +/-- 0.01

146 236 305

+ / - 31 +/-- 88 +/-- 42

72 48 51

1.13 1.60 2.30

+]-- 0.08 +/-- 0.05 +/ 0.19

+/

17.15

+/-- 3.9

213

+/

250

+/-- 25

85

1.35

+ / - 0.08

0.39

33

87 Table 4. Pigment content (I0 replicates), quantum yield and chlorophyll fluorescence (3 replicates) of soybean leaves grown under constant or fluctuating PFD ( + / - confidence interval with a probability of 95%). Irradiance #Mm - 2 s - I

Chlorophylls mgm 2

Chla/Chlb

Chl/Car

Quantum yield mMCO2M lphot.

Fluorescence reh Night

Light

(lOb) Constant 220 400 800

199 203 266

+ / - 18 + / - 35 + / - 60

3.6 3.8 4.2

+ / - 0.18 + / - 0.18 + / - 0.18

6.6 6.1 5.7

+ / 0.18 + / 0.54 + / - 0.34

9.4 18.2 35.5

+/+/ +/-

Fluctuating 400

168

+ / - 22

4.1

+/

5.3

+/

23.0

+ / - 0.55

0.18

0.54

1.3 1.1 1.3

94 94 100

83 78 91

79

62

is very low. It is clear that leaf pigements are affected by the very high transient PFD. But this bleaching is not high enough to affect the quantum yields. It must be noticed that the maximum quantum yield is rather low compared to generally reported values [7]. The leaf pigment content was not very high and the ratio of Chlorophyll/Carotenoid was probably not optimum [8]. It would not be due to a deficiency in the nitrogen nutrition: Rhizobium activity, estimated in the same experiment with the C2H2 assay was high enough to meet the plants nitrogen requirements (datas not shown). Plant submitted to transient illumination responded like photoinhibited plants. Indeed (a) leaf pigment content is decreased and (b) the yield of chlorophyll fluorescence was much more affected by a fluctuating light period of 10 hours and the dark recovery is not complete. Primary storage capacities of photosynthetic products in the leaf could influence the response to light fluctuation: under high PFD it has been shown that the triose phosphate utilization could be a limitation to the net CO2 uptake [26]. The excess of primary photosynthetic products, i.e. starch and sucrose, would be metabolized during the period of low light intensity. Table 5 shows t h e partitioning of carbon in leaves after 10 hours of light and 10rain of metabolism. Starch synthesis was slightly increased by high light. But the main primary photosynthetic product was soluble, and thus probably sucrose. Fluctuating Table 5. Storage of the primary photosynthetic products by soybean leaves grown under constant or fluctuating PFD after 10 hours of light (5 replicates, + / - confidence interval with a probability of 95%).

Irradiance (pM m 2 s ~)

% of radioactivity Soluble in 50% EtOH

Starch

Turn over rate of exportable radioactivity hours

Proteins and lipids

Constant 220 400 800

82 86 86

+/+/+/-

5.5 1.8 1.8

0.25 0.55 0.75

+/+/+/-

0.37 0.64 0.64

17.7 13.6 13.0

+/+/+/-

5.5 2.9 2.6

29 23 26

+/+/+/-

20 0 13

Fluctuating 400

83

+/-

5.5

0.45

+ / - - 0.39

15.5

+/-

6.4

21

+/-

3.7

88 conditions did not modify this pattern, except that the labelling o f the protein and lipid fraction increased probably because the turn-over o f this cellular fractions did increase. This interpretation can be related to the previously reported photoinhibition. U n d e r low light the relatively high p r o p o r t i o n o f radioactivity in the protein and lipid fraction could be attributed to the fact that under a limiting c a r b o n supply, more carbon is devoted to maintenance o f the photosynthetic cells and less is exported. The turn-over time o f exportable c a r b o n measured during 24 hours was high and did not depend on the P F D . U n d e r fluctuating light it was slightly decreased. In conclusion, fluctuating light conditions apply during the growth o f soybean leaves are almost equivalent to constant conditions except two m a j o r differences. Light adaptation is similar for most o f the studied characters, but there is found a significant change o f the activation rubisco in leaves adapted to the fluctuating regime. This effect does not affect net CO2 uptake. In addition the very high light flux applied transiently seems to affect the photochemical apparatus at least in the loss o f chlorophyll and fluorescence. The consequences of this photoinhibition are limited in soybean leaves since the photosynthetic q u a n t u m yield is not significantly affected. But the total biomass production after 60 days o f growth is 30% lower (data n o t shown). Accordingly photosynthetic capacities are p r o b a b l y not maintained during the whole p h o t o p e r i o d in the growth c h a m b e r with fluctuating P F D . Hence this adverse effect o f a transient high P F D is likely to be more significant for shade ecotypes.

References 1. Bj6rkman O (1973) Comparative studies on photosynthesis in higher plants. In: Giese AC (eds) Current Topics in Photobiology Photochemistry and Photophysiology, Vol. 8, p. 1-63. London/New York: Academic Press 2. Cammaert D and Jacob M (1980) A simple electrophoretic procedure for the determination of polypeptide composition of the subunits of fraction 1 protein. Anal Biochem 109:317 320 3. Caemmerer S Von and Farquhar GD (1981) Some relationships between the biochemistTyof photosynthesis and the gaz exchange of leaves. Planta 153:376-387 4. Farquhar GD and Caemmerer S Von (1982) Modeling of photosynthetic response to environmental conditions. In: Lange OL, Nobel PS, Osmond CB and Ziegler H (eds) Physiological Plant Ecology. II. Water Relations and Carbon Assimilation. Encycl. Plant Physiol., New Ser., Vol. 12B, pp 549-587. Berlin: Springer-Verlag 5. Gaudillere JP (1974) Amelioration du dosage spectrophotom+trique des chlorophylles a et b et des carot6noides dans les extraits foliaiers. Physiol Veg 12:595-599 6. Gaudillere JP (1977) Effect of periodic oscillations of artificial light emission on photosynthetic activity. Physiol Plant 41:95 98 7. Gaudillere JP (1979) Variabilit6 du rendement quantique de la photosynth6se des plantes sup~rieures. Th~se Doc. es Sciences. Universit6 Paris-Sud. Centre d'Orsay 8. Gaudillere JP (1979) Effet des carotdnoides sur le rendement quantique de la photosynth6se chez Tritieum aestivum. Physiol Veg 17:777 787 9. Gross LJ and Chabot BF (1979) Time course of photosynthetic response to change in incident light energy. Plant Physiol 63:1033 1038 10. Gutteridge S, Parry MAJ, Burton S, Keys AJ, Mudd A, Feeney J, Servaites JC and Pierce J (1986) A nocturnal inhibitor of carboxylation in leaves. Nature 324:274-276

89 ll. 12. 13.

14.

15. 16.

17. 18.

I9. 20. 21. 22. 23. 24. 25. 26.

27. 28.

29. 30.

31.

Inskeep WP and Bloom PR (1985) Extinction coefficients of chlorophyll a and b in N,NDimethylformamide and 80% acetone. Plant Physiol 77:483-485 Kalia VC and Drevon JJ (i985) Variation of nitrogenase activity (C2H2) reduction during in situ incubation of root nodules of Glycine M a x L. Merr. CR Acad Sc Paris 301:591-596 Melis A and Harvey GW (1981) Regulation ofphotosystem stoechiometry, chlorophyll a and chlorophyll b content and relation to chloroplast ultrastructure. Biochim Biophys Acta 637: 138-145 Morgan CL and Austin RB (1986) Analysis of fluorescence transients of DCMU-treated leaves of Triticum species to provide estimates of the densities of photosystem II reaction centers. Photosynth Res 7:203 219 Pearcy RW, Osteryoung K and Calkin HW (1985) Photosynthetic responses to dynamic light environments by Hawaiian trees. Plant Physiol 79:896-902 Perchorowicz JT, Raynes DA and Jensen RG (1982) Measurement and preservation of the in vivo activation of Ribulose 1,5-Bisphosphate carboxylase in leaf extracts. Plant Physiol 69: 1165-1168 Pollard DFW (1970) The effects of rapidly changing light on the rate of photosynthesis in largetooth aspen (Populos grandidentata). Can J Bot 48:823-829 Prioul JL (1971) R4action des feuilles de Lolium multiflorurn fi l'4clairement pendant la croissance et variation des r6sistances aux 6changes gazeux photosynth4tiques. Photosynthetica 5:364-375 Rabinowich El (1956) Photosynthesis and Related Processes. Vol. II Part 2. New York: Interscience Rygiewicz PT, Bledsoe CS and Glass AD (1984) A comparison of method for determining compartimental analysis parameters. Plant Physiol 76:913 917 Servaites JC and Torisky RS (1984) Activation state of ribulose bisphosphate carboxylase in soybean leaves. Plant Physiol 74:681-686 Servaites JC (1985) Binding of a phosphorylated inhibitor to ribulose bisphosphate carboxylase/oxygenase during the night. Plant Physiol 78:839 843 Scholender PF, HammeI HT, Bradstreet ED and Hemingsen EA (1965) Sap pressure in vascular plants. Science 148:339-346 Sharkey TD (1985) Photosynthesis in intact leaves of C 3 plants: physics, physiology and rate limitation. Bot Rev 51:53-105 Sharkey TD, Berry JA and Raschke K (1985) Starch and sucrose synthesis in Phaseolus vulgaris as affected by light, CO 2 and abscissic acid. Plant Physiol 77:617 620 Sharkey TD, Stitt M, Heineke D, Gerhart R, Raschke K and Heldt HW (1986) Limitation of photosynthesis by carbon metabolism. II. 02-insensitive CO 2 uptake results from limitation of triose phosphate utilization. Plant Physiol 81:1123 1129 Thornly JHM (1974) Light fluctuations and photosynthesis. Ann Bot 38:363 373 Torisky RS and Servaites JC (1984) Effect of irradiance during growth of Gl)'cine max on photosynthetic capacity and percent activation of ribulose. 1,5-bisphosphate carboxylase. Photosynth Res 5:251-261 Turner NC (198I) Techniques and experimental approaches for the measurement of plant water status. Plant and Soil 58:339-366 Vu NC, Allen LH and Bowes G (1983) Effects of light and elevated atmospheric CO 2 on the ribulose bisphosphate carboxylase activity and ribulose bisphosphate level of soybean leaves. Plant Physiol 73:729 734 Vu CV, Bowes G and Allen LH (1986) Properties of ribulose 1,5-bisphosphate carboxylase from dark and light exposed soybean leaves. Plant Physiol 44:119 123