The Synthesis of [14c]Starch from [14~]~ucrose in Isolated Wheat Grains is Dependent upon the Activity of Soluble Starch Synthase

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1 Aust. J. Plant Physiol., 1993, 20, The Synthesis of [14c]Starch from [14~]~ucrose in Isolated Wheat Grains is Dependent upon the Activity of Soluble Starch Synthase C. F. Jennerq K. SiwekA and J. S. HawkerB A Department of Plant Science, The University of Adelaide, Waite Agricultural Research Institute, PMB 1, Glen Osmond, SA 5064, Australia. Department of Botany, The University of Adelaide, GPO Box 498, Adelaide, SA 5001, Australia; died 9 March Abstract Soluble starch synthase activity decreased in isolated wheat grains heated in vials at temperatures between 31 and 40 C; a decrease of more than 50% occurred at 35'C within 30 min. Sucrose synthase activity was not significantly affected by heating and ADPglucose pyrophosphorylase decreased to a lesser extent and more slowly than soluble starch synthase. Wheat grains which were heated for 1 h at 35 C and allowed to recover at 20 C exhibited partial recovery of soluble starch synthase activity within 4 h. These responses in isolated grains to heating resembled effects reported elsewhere in intact ears. Isolated grains were exposed to a range of temperatures for 1 h prior to incubation in ['4C]sucrose at 20 C. Within the range C, the effect of pretreatment temperature on the activity of soluble starch synthase was highly correlated with the incorporation of I4c into starch. Control coefficients close to unity indicated that the activity of soluble starch synthase imposed a high degree of control on the rate of starch synthesis in this system. These results with isolated grains support a previous suggestion that the activity of soluble starch synthase at elevated temperature in wheat is an important factor limiting the accumulation of dry matter through effects on the synthesis of starch. Introduction Exposure of wheat ears to high temperature (in the range C) decreases the conversion of sucrose to starch in the developing wheat endosperm (Bhullar and Jenner 1986). More recent work suggests that this effect is due to a reduction in the amount of soluble starch synthase activity in the endosperm at elevated temperature (Hawker and Jenner 1993). Heating ears for 1 day at 35 C reduced the activity of soluble starch synthase by almost 50%. Additional heating for 3-9 days reduced activity a little more, to about 40% of the values recorded for unheated (21 C) ears. In ears that had been heated for 2, 4, or 7 days and then returned to lower temperature, soluble starch synthase activity nearly recovered to levels observed in ears that had not been heated. Although the experimentally imposed environmental conditions in the work described above (Hawker and Jenner 1993) reflect those prevailing in the field, the heating procedure is not convenient for investigating the effects of temperature on starch synthesis at the physiological and biochemical levels. Temperature of the grain itself is difficult to measure in situ and precise control of temperature is not easily achievable. Accordingly, a system of heating grains after isolating them from the ear was developed, and this system has been used for comparing the effects of temperature on the rates of incorporation of 14c from [14~]sucrose into starch with soluble starch synthase activity. The results provide further support for the view that elevated temperature reduces starch synthesis through its effects on soluble starch synthase activity, and implicate this enzyme as a cardinal factor controlling starch accumulation in developing wheat endosperm /93/030329$05.00

2 C. F. Jenner et al Materials and Methods Wheat (Triticum aestivum L. cw. Sun 9E, Trigo 1 and Lyallpur) was grown as described by Jenner (1991). Sucrose synthase (EC ) and ADPglucose pyrophosphorylase (EC ) were assayed as described by Hawker and Jenner (1993). Soluble starch synthase (EC ) was assayed using the anion exchange procedure for removing unreacted [14~]~~~glucose, and granule bound starch synthase was assayed using the methanol-kc1 method; both methods are described by Hawker and Jenner (1993). Heating Isolated Grains Five wheat grains (16-23 days after anthesis) were removed from the two most proximal florets of spikelets in the middle of the ear and incubated intact in 5 ml scintillation vials lined with moist Whatman No. 1 filter paper and capped with non-absorbent cotton wool. The vials were placed in water baths at temperatures between 20" and 40 C. In recovery experiments, vials were held at 3S C and then returned to a water bath at 20 C for specified periods. At intervals grains were removed, blotted with absorbent paper, peeled by removing the outer pericarp, the tissues of the furrow and the embryo, and the endosperm was homogenised for assay of enzyme activities. Comparison of Incorporation into Starch and Soluble Starch Synthase Activity Grains of wheat (17 to 18 days after anthesis) were heated in vials at 20, 31, 35 and 38 C as described above. After 1 h the grains were peeled and transferred to two sets of 25 ml conical flasks containing 2.5 ml of 146 mm sucrose solution. The flasks were loosely plugged with non-absorbent cotton wool and gently shaken for 2 or 4 h at 20 C. One set also contained 1 pci of [14~]sucrose per flask. The grains from this set were washed with 20 ml of H20 on a small strainer, blotted and heated at 80 C in 80% ethanol for 15 min. The radioactivity in the starch and in the soluble sugar was determined as described by Bhutlar and Jenner (1986). The amount of starch synthesised was calculated using the specific radioactivity of the sucrose supplied. Grains from the non-radioactive set were extracted and assayed for soluble starch synthase. Replication The results shown in Figs 1-4 are means of three replicate ears. Those in Figs 5 and 6 are means of five experiments. Vertical bars shown on the figures are 1.s.d.s (PGO.05). Results Heating isolated but otherwise intact grains in vials in a humid atmosphere for 1 h at 31 C and above caused a decrease in soluble starch synthase activity (Fig. 1). At 40 C there was a drop in activity of almost 70%. Compared with grains assayed immediately upon Fig. 1. The activity of soluble starch synthase in wheat grains of cv. Sun 9E removed from the ear 23 days after anthesis and heated for 1 h in vials in a moist atmosphere. Data are expressed as % of the activity in grains kept at 20 C which was 3.6 nmol (grain min)-i. The vertical bars in this and subsequent figures are 1.s.d.s (PGO.05). Pretreatment temperature ( OC)

3 Starch Synthesis and Soluble Starch Synthase in Wheat removal from the ear, grains maintained at 20 C for 1 h did not lose any of their soluble starch synthase activity. Granule bound starch synthase activity was not significantly affected by these heating treatments. In one experiment (grains isolated on day 18 after anthesis), the value for granule bound activity after 1 h of heating at 35 C was 97'70 of the value observed at 21 C (mean of four replicates). A time course of heating at 35 C showed that heating for only 30 min caused considerable reduction of soluble starch synthase activity. Further heating caused a greater reduction (Fig. 2), but the effects of heating diminished with time. After 2 h, ADPglucose pyrophosphorylase activity had decreased significantly, but the rate of decrease over this period was much less than that of soluble starch synthase. Sucrose synthase activity dropped by only lo%, a non-significant effect, in 4 h. Fig. 2. The activities of three enzymes in wheat grains of cv. Sun 9E, removed from the ear 16 days after anthesis, which had been heated at 35'C in vials for up to 4 h; data are expressed as % of the initial activity. Initial activities of sucrose synthase (e), ADPglucose pyrophosphorylase (m) and soluble starch synthase (0) were 62, 69 and 7.8 nmol (grain rnh- ' respectively Time of heating (h) In a recovery experiment, grains were heated for 1 h at 35 C and then transferred to a temperature of 20 C for up to 4 h. Soluble starch synthase activity decreased after 1 h at 35 C to less than 50% of the original value, but transfer to 20 C arrested the decline in activity and some recovery was observed after 4 h (Fig. 3b). Heating for 1 h had little effect on ADPglucose pyrophosphorylase activity, and there was no change in activity after transfer to 20 C (Fig. 3a). Responses to heating were compared in two other cultivars: Trigo 1 and Lyallpur (Fig. 4). Based on their grain-filling responses to temperature (Wardlaw et al. 1989), Trigo 1 appears more tolerant of high temperature than Lyallpur. Soluble starch synthase activity was decreased by heating in both cultivars, a greater effect being observed at 38 C than at 32 C (Fig. 4). ADPglucose pyrophosphorylase and sucrose synthase showed only minor or no changes in activity, and there were no significant differences between the two cultivars. Evidently, differences between these two cultivars in their sensitivity to high temperature are not due to differential responses to temperature in the activity of these enzymes under these conditions. In experiments designed to evaluate the relationships between the rate of starch synthesis and soluble starch synthase activity, grains were pre-treated for 1 h in vials at different temperatures and the subsequent incorporation of 14c from [ 1 4 ~ ] ~ into ~ starch ~ r ~ at ~ 20 C e was compared with the activity of soluble starch synthase. Similar amounts of radioactivity were extracted in hot ethanol from the cultured grains pre-treated at all temperatures (results not shown) indicating that the uptake of [14~]sucrose was not affected by the temperature of the pre-treatment. Over 4 h the rate of incorporation of 14c into starch at 20 C was

4 C. F. Jenner et al. E d C I (a) =.- (b) I I, Time after excision (h) Time after excision (h) Fig. 3. Effect of transferring wheat grains to a temperature of 20 C after heating them in vials for 1 h at 35OC on the activities of ADPglucose pyrophosphorylase (a) and soluble starch synthase (b). Data are expressed as % of initial values which were (a) 63 and (b) 5.3 nmol (grain min)-l. Grains were removed from the ear of cv. Sun 9E 16 days after anthesis. Grains were maintained at 20 C throughout (0) or heated for 1 h (0) and then transferred to 20 C for 1 to 4 h. There were no significant differences in (a). Fig. 4. The activities of sucrose synthase (0) ADP glucose pyrophosphorylase (0) and soluble starch synthase (B) in grains of cv. Trigo 1 (-) and cv. Lyallpur (------) which had been heated in vials for 100 min. Grains were removed from the ear at 16 days after anthesis. Data are expressed as % of the activity in grains kept at 20 C. At 20 C, average values for the three enzymes and two cvv. respectively were 51 and 61, 65 and 60, 6.9 and 6.1 nmol (grain min)-'. Only starch synthase activities were significantly affected by temperature Pretreatment temperature ("C) essentially linear with time, but incorporation was lower at 35OC and at 38 C (Fig. 5). The rates of starch synthesis over the 2 to 4 h period showed dependence on pretreatment temperature as did the means of the soluble starch synthase activities assayed at 2 and 4 h (Fig. 6). The activity of soluble starch synthase, which was assayed at 30 C, was more than 3-fold greater than the calculated rates (2 to 4 h) of starch synthesis which were measured at a temperature of 20 C. Sufficient sucrose (non-radioactive) was present in the endosperm at the beginning of the incubation to support starch synthesis for just over 3 h (Ugalde and Jenner 1990). Uptake of [14~]sucrose during the incubation would have maintained the size of this pool (see Jenner and Rathjen 1977). After 4 h of incubation, the specific radioactivity of sucrose in the endosperm would have been about 75% (Jenner 1974) of the value of the sucrose supplied. Moreover, the amount of radioactivity in the soluble sugars did not change between 2 and 4 h of incubation implying that the pool of sucrose had reached near constant specific activity before 2 h. If the temperature difference (referred to above) and

5 Starch Synthesis and Soluble Starch Synthase in Wheat Fig. 5. The accumulation of radioactive starch produced from ['4~]sucrose by isolated grains of cv. Sun 9E (sampled 17 days after anthesis), which had been pre-treated by heating for 1 h in vials at the temperatures indicated, prior to incubation at 20 C with [14~]sucrose. Time (h) Fig. 6. Effects of pre-treatment temperature on (a) rates of starch synthesis between 2 and 4 h after the beginning of incubation in [14~]sucrose, and (b) means of the 2 and 4 h values of the activities of soluble starch synthase. Further details are in the legend to Fig. 5. dilution (to 75%) of the supplied ['4~]sucrose are taken into account, the actual rate of starch synthesis was probably much closer to the activity of soluble starch synthase than the measured values suggest. Discussion The results of this paper confirm the conclusion reached in earlier work that the activity of soluble starch synthase in wheat grains is very sensitive to heat treatment. Whether the grains are heated in situ (Hawker and Jenner 1993) or in vitro (this paper), the decrease in soluble starch synthase is accompanied by a similar decrease in dry matter accumulation or in starch synthasis as measured by incorporation of 14c from supplied [14~]sucrose. The current results generally are in accordance with the earlier finding of Rijven (1986) that soluble starch synthase activity was decreased in heat treated wheat grains. However, the use of an assay method for soluble starch synthase (Hawker and Jenner 1993) that provides reliable estimates of maximum catalytic activity in wheat endosperm, has focused attention on the closeness of the association between starch synthesis and soluble starch synthase activity in the developing grain. Responses to heating of grains isolated from the ear show several features in common with effects of heating in other experimental systems. For example, Rijven (1986) observed

6 C. F. Jenner et al. that soluble starch synthase activity was reduced substantially by 1 h prior exposure of slices of grain to temperatures above 30 C. In whole isolated grains (Fig. 2) the response to heating at 35 C is clearly evident after 0.5 h. Although no significant reduction in soluble starch synthase activity was detected in grains taken from ears which had been heated at 35 C for 4 h (results not shown), Rijven (1986) reported that soluble starch synthase activity was reduced by about 60% 0.5 h after intact plants were transferred from 21 to 37 C. Recovery, or partial recovery, of soluble starch synthase activity is observable in isolated grains (Fig. 3b) after they had been transferred from 35 to 20 C, and also after ears (Hawker and Jenner 1993) or whole plants (Rijven 1986) were transferred from warm to cool conditions. Finally, the time course of loss of activity at high temperature does not follow first order kinetics in isolated grains (Fig. 2) or in grain slices (Rijven 1986), or in heated ears (Hawker and Jenner 1993) or intact plants transferred to high temperature (Rijven 1986). Instead, an initial rapid decline in activity is followed by a more gradual loss implying more than one action of heat and/or multiple forms of soluble starch synthase differing in their sensitivity to high temperature. Thus, the responses observed in the isolated grain system appears to resemble closely effects of heat on starch accumulation and enzyme activity in vivo as well as in other in vitro systems. Heating intact ears had little effect on the activity of granule bound starch synthase and no significant effects were observed on the activity of branching enzyme, or on amylase activity (Hawker and Jenner 1993). There was a negligible effect of heating isolated grains on the activity of granule bound starch synthase, sucrose synthase, and ADPglucose pyrophosphorylase activity was not affected at all (Fig. 3a), or was reduced after 1 h of heating at 35 C by only 9.7% (Fig. 2). Moreover, maximum catalytic activity of ADPglucose pyrophosphorylase was approximately 10-fold greater than the observed rate of starch synthesis. It is reasonable to conclude, therefore, that the reduction in soluble starch synthase activity alone can explain the reduction in the synthesis of I4c starch (Fig. 6). Plotting the fractional change in the rate of starch synthesis against the fractional change in activity of soluble starch synthase (a flux-control plot, see Kacser and Burns 1973) indicates a near-linear relationship (Fig. 7), with a slope of unity. Alternatively, assuming a hyperbolic relationship, where the slope becomes steeper as the activity of the enzyme decreases, the flux control coefficient can be estimated (e.g. Neuhaus and Stitt 1990) using the equations developed by Torres et al. (1986). Taking the data for 20 and 31 C, and for 20 and 3S C, these estimates are 0.88 and 1.22 respectively which values also imply that the true value of the flux control coefficient is unity or not far removed from unity. The simplest interpretation of these results is that the activity of soluble starch synthase imposes a high degree of control over the synthesis of starch at the mid grain filling stage Fig. 7. Control plot of the effects of heating wheat grains on starch synthesis and soluble starch synthase activity. Data are calculated from Fig. 6. I Starch synthase actlvity (% of 20'~ grains)

7 Starch Synthesis and Soluble Starch Synthase in Wheat of development in this isolated grain system. In light of the proposal (Hawker and Jenner 1993) that lowered dry matter accumulation at elevated temperature is primarily due to a reduction in the activity of soluble starch synthase, the present findings support the view that, of all of the enzymes in the sucrose-to-starch pathway considered in this work, it is the activity of soluble starch synthase which is the most important in limiting starch accumulation (in the grain) and thus the yield of wheat. Whether or not other enzymes contribute significantly to control at other stages of development remains to be established. Acknowledgments This work was supported by a grant from the Australian Research Council. Helpful comments by Dr K. Denyer were appreciated. The technical assistance of Mr R. Batt and the assistance of Ms J. Guerin and Ms H. Weston with the preparation of this manuscript are gratefully acknowledged. References Bhullar, S. S., and Jenner, C. F. (1986). Effects of temperature on the conversion of sucrose to starch in the developing wheat endosperm. Australian Journal of Plant Physiology 13, Hawker, J. S., and Jenner, C. F. (1993). High temperature affects the activity of enzymes in the committed pathway of starch synthesis in developing wheat endosperm. Australian Journal of Plant Physiology 20, Jenner, C. F. (1974). An investigation of the association between the hydrolysis of sucrose and its absorption by grains of wheat. Australian Journal of Plant Physiology 1, Jenner, C. F. (1991). Effects of exposure of wheat ears to high temperature on dry matter accumulation and carbohydrate metabolism in the grain of two cultivars. I. Immediate responses. Australian Journal of Plant Physiology 18, Jenner, C. F., and Rathjen, A. J. (1977). Supply of sucrose and its metabolism in developing grains of wheat. Australian Journal of Plant Physiology 4, Kacser, H., and Burns, J. A. (1973). The control of flux. Symposia of the Society for Experimental Biology 27, Neuhaus, H. E., and Stitt, M. (1990). Control analysis of photosynthate partitioning. Impact of reduced activity of ADPglucose pyrophosphorylase or plastid phosphoglucomutase on the fluxes to starch and sucrose in Arabidopsis thaliana (L.) Heynh. Planta 182, Rijven, A. H. G. C. (1986). Heat inactivation of starch synthase in wheat endosperm tissue. Plant Physiology 81, Torres, N. U., Mateo, F., Melendez-Hevia, E., and Kacser, H. (1986). Kinetics of metabolic pathways. A system in vivo to study the control of flux. Biochemical Journal 234, Ugalde, T. D., and Jenner, C. F. (1990). Substrate gradients and regional patterns of dry matter deposition within developing wheat endosperm. I. Carbohydrates. Australian Journal of Plant Physiology 17, Wardlaw, I. F., Dawson, I. A., and Munibi, P. (1989). The tolerance of wheat to high temperatures during reproductive growth. 11. Grain development. Australian Journal of Agricultural Research 40, Manuscript received 21 December 1992, accepted 12 March 1993