Changes in the activity of sucrose-synthesizing enzymes in developing leaves of Lolium temulentum

Plant Science Letters, 7(1976)27--31 © ElsevierScientific Publishing Company, Amsterdam -- Printed in The Netherlands

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CHANGES IN THE ACTIVITY OF SUCROSE-SYNTHESIZING ENZYMES IN DEVELOPING LEAVES OF LOLIUM T E M U L E N T U M

CHRISTOPHER JOHN POLLOCK Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, SY23 3EB (Great Britain) (Received February 9th, 1976) (Accepted March 11th, 1976)

SUMMARY Sucrose phosphate synthetase was assayed throughout the development of t h e fourth leaf of Lolium temulentum L. Activity was maximal at the time of ligule formation and subsequently declined. Specific sucrose phosphate phosphatase was always present in excess although sucrose synthetase activity was considerably lower and remained unchanged. Maximum levels of sucrose phosphate synthetase activity are comparable in magnitude to levels of photosynthetic activity in mature grass leaves and with estimates of sucrose transport from the flag leaf into developing grass seeds.

INTRODUCTION It has been proposed that sucrose phosphate synthetase (EC 2.4.1.14) is the principal enzyme of sucrose synthesis in plants, and that sucrose synthetase (EC 2.4.1.13) is primarily an enzyme of sucrose cleavage [1]. If this is so the activity of sucrose phosphate synthetase represents the maximum capacity of a photosynthesizing leaf to export sucrose. Estimates of the activity of sucrose phosphate synthetase have been made in the leaves of a variety of plants [ 2 - 5 ] . Marked variation exists between species and between plants of the same species grown under different conditions [3]. In general the activities of sucrose phosphate synthetase are substantially higher than those of sucrose synthetase. During the normal development of individual leaves there is a transition from a net input to a net export of photosynthate [6]. This investigation was carried out to estimate the maximum catalytic activities [7] of sucrose phosphate synthetase and sucrose synthetase throughout the development of the fourth leaf of Lolium temulentum (L.). These activities were compared with

28 the known rates of photosynthesis in grass leaves and estimates of the likely rates of movement of sucrose out of individual leaves. In order to obtain reliable estimates of maximum catalytic activity care was taken to minimize losses during extraction and to ensure that the assay methods used were appropriate to the tissue involved [4,8]. MATERIALS AND METHODS Plants of Lolium temulentum L. Ba 3081 (summer annuM) were grown from seed in John Innes No. 1 compost in a controlled environment room. The photoperiod was 8 h and the day/night temperatures 20°C and 15°C, respectively. Light intensity at the plant surface was 100 W/m s. Prior to ligule formation whole leaves were harvested but, subsequently, leaf blades only were used. Extracts for assays of sucrose phosphate synthetase and sucrose phosphate phosphatase were prepared as described by Lyne and ap Rees [9]. Sucrose synthetase was extracted and concentrated as described by Pressey [10], except that the material was extracted in 4 vol. of 0.1 M phosphate buffer pH 8.0 containing 0.1 M NaC1 and 0.01 M cysteine. The fraction of this extract which precipitated between 30 and 60% saturation by ammonium sulphate was resuspended in 1 ml of buffer and assayed after overnight dialysis against extraction buffer at 4°C. Sucrose phosphate synthetase was assayed either by the method of Lyne and ap Rees [9] or by that of Bird et ai. [4]. In the latter case the reaction mixture contained 4 gmoles of UDPG and 6 ~moles of [U-14C]fructose-6phosphate (0.08 gCi) in a total volume of 0.1 ml. Sucrose phosphate phosphatase was assayed by the method of Hawker and Hatch [11] using glucosyl-[ U-14C]fructosyl-6-phosphate prepared by the method of Hatch [12]. Sucrose synthetase was assayed as described by Lyne and ap Rees [9], except that the reaction mixture contained 10 gmoles sucrose, 2.5 #moles UDPG, 3.0 gmoles [U-14C]fructose (0.08 gCi) and 6.6 gmoles Tris--HC1 at pH 7.5 in a final volume of 0.1 ml. In all cases sucrose was separated by paper chromatography and counted on paper by liquid scintillation spectrometry at 48% efficiency. Chlorophyll and soluble protein were assayed as described previously [ 13 ]. RESULTS AND DISCUSSION Sucrose phosphate synthetase has been assayed by measuring the amount of sucrose phosphate formed under conditions where hydrolysis by sucrose phosphate phosphatase was inhibited. Bird et ai. [4] claim that better estimates can be obtained by coupling the reaction to sucrose phoaphate phosphatase which was presumed to be in excess, and measuring U D t ~ and F-6-P dependent sucrose synthesis. Both methods were teited on leaves of L. temulentum harvested at three different growth stages. Measurements were made of sucrose phosphate phosphatase activity to determine whether

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or not it was present in excess. The results are given in Table I and show that higher activities were obtained using the coupled reaction, and that in all cases the rates were limited by the activities of sucrose phosphate synthetase, not sucrose phosphate phosphatase. Hydrolysis of F-6-P under the same conditions was less than 5% of that of sucrose phosphate. It is proposed that the coupled assay can be used where non-limiting sucrose phosphate phosphatase activity has been demonstrated together with low levels of non-specific phosphatases. Individual components of the reaction mixtures of both sucrose phosphate synthetase and sucrose synthetase were varied to give optimum rates. Losses of activity during extraction were investigated by determining the levels of both enzymes in tissue of different ages and mixtures of tissues as described previously [8]. For both enzymes, the activities recovered from the mixed samples were within 10% of those predicted from the measurements made on the separate samples. Using these extractions and assay conditions, estimates of the activities of sucrose synthetase and sucrose phosphate synthetase were made at various times during the development of the fourth leaf. The results are shown in Table II. The activities of sucrose synthetase are very much lower than those of sucrose phosphate synthetase and remain relatively constant. This provides further evidence against a synthetic role in leaves for sucrose synthetase. Sucrose phosphate synthetase activity rises to a maximum of around 10 ~moles/g fr. wt/h at the time of ligule formation and subsequently declines at about the same rate as leaf soluble protein. Similar patterns of activity have been obtained for several enzymes of intermediate metabolism in developing grass leaves [ 14], and gas exchange also declines immediately after full leaf expansion [15]. The maximal values for sucrose phosphate synthetase activity were compared with estimates of photosynthetic rates and sucrose transport derived from the literature in order to determine whether metabolic control of sucrose TABLE I COMPARISON OF ASSAY METHODS F O R SUCROSE PHOSPHATE SYNTHETASE IN EXTRACTS OF LEAVES OF L. T E M U L E N T U M HARVESTED AT D I F F E R E N T TIMES A F T E R EMERGENCE OF THE L E A F TIP Days after

Enzyme activities (~mole product/g ft. wt/h) a

emergence

of the leaf tip

Sucrose phosphate b synthetase ( 1 )

Sucrose phosphate c synthetase (2)

Sucrose phosphate phosphatase

2 12 24

3.4 6.5 2.1

4.5 8.9 3.0

180 396 215

aAll values are the mean of five replicate assays. bAssay 1 by the method of Lyne and ap Rees [ 11 ]. CAssay 2 by the m e t h o d of Bird et al. [4].

30 TABLE II CHANGES IN THE ACTIVITIES OF SUCROSE PHOSPHATE SYNTHETASE AND SUCROSE SYNTHETASE DURING THE DEVELOPMENT OF THE F O U R T H L E A F OF L. TEMULENTUM Days after emergence of the leaf tip

0 4 7 11 b 14 21 c 28

Enzyme activities a (~mole product/g fr. wt/h) Sucrose phosphate synthetase

Sucrose synthetase

4.3 5.9 8.5 9.6 9.0 5.3 1.4

0.5 N/A N/A 0.4 N/A N/A 0.6

Soluble protein (mg/g ft. wt)

Chlorophyll (mg/g fr. wt)

9.4 11.0 13.2 13.0 12.5 9.8 5.9

0.4 0.9 1.2 1.3 1.5 1.3 0.8

aAll values are the mean of four replicate assays. bLigule fully formed. c Onset of yellowing. N/A, Not assayed.

production by this reaction was feasible. Observed rates of net gas exchange in mature leaves of Lolium spp. were in the range of 0.3--0.5 mg CO2/m2/s [16]. Specific leaf weight values for L. temulentum were approximately 300 g fr.wt/m 2, which gives values of 7--11 ~moles sucrose synthesized/g ft. wt/h if all the CO2 fixed was synthesized into sucrose. It is more difficult to obtain estimates of the amount of sucrose exported from individual leaves. Estimates of grain filling can be used but are complicated by the contribution of ear photosynthesis. It has been proposed that in wheat about 25% of the grain carbon is provided by ear photosynthesis during the period of maximum grain filling and that the remainder comes from flag leaf photosynthesis [17]. Mature flag leaves of L. temulentum weigh about 180 mg and hence would be capable of exporting up to 40 ~moles of sucrose per day. Ears of plants grown under these conditions contain about 30 grains and during the first 10 days after anthesis mean dry weight gains of about 0.5 rag/grain/day were recorded. This is equivalent to 40--50 ~moles sucrose per day, of which 30--40/~moles would be provided by the flag leaf. The values assigned to these processes must be regarded as approximate, but it appears that the maximum catalytic activity of sucrose phosphate synthetase is of the same magnitude as the processes which provide the precursors for sucrose synthesis and which determine its ultimate fate. It is significant that higher values for sucrose phosphate synthetase have been obtained from leaves of C-4 grass species where leaf photosynthetic rates are also higher [ 5]. Further experiments will be necessary to determine whether

31 sucrose p h o s p h a t e s y n t h e t a s e a c t i v i t y limits sucrose e x p o r t a t a n y stage o f leaf d e v e l o p m e n t . ACKNOWLEDGEMENTS I wish t o t h a n k P r o f e s s o r J.P. C o o p e r , D i r e c t o r o f t h e Welsh P l a n t B r e e d i n g S t a t i o n , f o r his e n c o u r a g e m e n t d u r i n g t h e c o u r s e o f t h e s e studies, a n d Miss J o a n Davies f o r v a l u a b l e t e c h n i c a l assistance. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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