Planta ,1990)181 : 104-108

P l a n t a 9 Springer-Verlag1990

Starch synthesis in developing wheat grain The effect of light on endosperm starch synthesis in vitro and in vivo G.A. Foxon, L. Catt, and P.L. Keeling* ICI Agrochemicals, Jealotts Hill Research Station, Bracknell, Berkshire, RGI 2 6EY, UK

Abstract. The effect of light on the in-vivo rate of starch synthesis in the endosperm of developing wheat (Tritic u m a e s t i v u m cv. Mardler) grain was studied. Individual grains from spikelets grown on the same spike either in darkness or bright light showed no difference in their ability to accumulate radioactivity or to convert this to starch over a 14-h period. Similarly, there was no difference in final grain dry weight between spikes which had been kept in either darkness or normal light from 10 d post anthesis. In contrast, when "half-grains" (grain which had been bisected longitudinally along the crease region) were incubated by being submerged in culture solution (in vitro) the incorporation of [14C]sucrose into starch was stimulated by increased irradiance. Further experiments showed that the in-vitro dependence on light could be linked to the availability of oxygen. We suggest that in vitro the diffusion of oxygen into the endosperm cells combined with an increased rate of respiration of the tissue during the incubation causes this limitation. Thus the dependence of starch synthesis on light is an artefact of the in-vitro incubation system. The photosynthetic ability of the green pericarp tissue can be used to prevent the development of anoxia in the endosperm tissue of half-grains incubated in vitro. In conclusion, we propose that starch synthesis in vivo is not dependent on oxygen production by photosynthesis in the green layer of the pericarp.

Key words: Endosperm - Grain filling

Pericarp - Photosynthesis - Starch synthesis - T r i t i c u m (starch synthesis)

containing layer of pericarp tissue. During most of the grain-filling period the pericarp tissue is green and capable of producing oxygen by photosynthesis (Nutbeam and Duffus 1978). Furthermore, because the outer pericarp tissue is relatively impermeable to carbon dioxide (Radley 1976) and to oxygen (Nutbeam and Duffus 1978) it has been suggested that a major function of the green layer may be the provision of oxygen to maintain starch synthesis in the inner endosperm tissue (Duffus 1979; Cochrane and Duffus 1979). Thus it was postulated than the rate of starch deposition in cereal endosperm is regulated, in part, by oxygen derived from green-layer photosynthesis. This attractive hypothesis received some support following the observation that [14C]sucrose incorporation into starch by grain incubated in vitro is sustained by the photosynthetic production of oxygen in the green layer of the pericarp (Gifford and Bremner 1981). These authors suggested that this light-oxygen effect provides a plausible explanation for the shell structure of wheat starch granules, which appears to correlate with the number of light-dark cycles during grain development (Buttrose 1962). We have examined the effect of light on starch synthesis in developing wheat grain under both in-vivo and in-vitro incubation conditions. Our aim was to establish whether or not starch synthesis in vivo was limited by the availability of oxygen derived from pericarp photosynthesis.

Material and methods Introduction

Plant material. A continuous supply of developing wheat (Triticum aestivum cv. Mardler, National Seed Development Organisation,

The developing wheat grain contains an inner starchstoring endosperm surrounded by an outer chlorophyll-

Newton, Cambridge, UK) grain was achieved by the following procedure: approx. 40 seeds were grown each week in John Innes No. 3 compost. After one week in a greenhouse the seedlings were moved to a cold room for vernalisation for six weeks at 4~ C with light from fluorescent lamps for 8 h per day. The vernalised seedlings were transplanted, two per pot, into 15-cm plant pots and returned to the greenhouse. Supplementary lighting was supplied from September until April for 16 h per day using sodium-vapor

* Present address: ICI Seeds, Garst Seeds Research Department, P.O. Box 500, Slater, IA 50010, USA Abbreviations: DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethylurea; dpa = days post anthesis; PCA = perchloric acid

G.A. Foxon et al. : Starch synthesis in developing wheat grain lamps; giving photosynthetically active radiation of approx. 500 p.mol p h o t o n s . m - 2 . s ~ (measured using a Crump quantum photometer; Crump Instruments, Billericay, Essex, UK). Fungal disease was controlled with Milgo fungicide (ICI, Fernhurst, Surrey, UK) and insect pests were controlled with Fumite DDT/Lindane insecticide generators (Pains-Wessex, Salisbury, Wiltshire, UK). After the emergence of the third leaf, the plants were fed each week with 0.1 g per pot Phostrogen fertiliser (Phostrogen, Corwen, Clwyd, UK). The developing ears were tagged with the date of anthesis.

Chemicals. [U-14C]Sucrose ( > 13 GBq-mmol 1, 350 mCi. mmol -~) was purchased from Amersham International PLC (Amersham, Bucks, UK). All other chemicals were obtained from either Sigma Chemical Co. (Poole, Dorset, UK) or BDH Chemicals (Poole, Dorset, UK). In-vivo experiments. Greenhouse-grown plants were transferred at 20 d post anthesis (dpa) to a growth cabinet (Fisons, Loughborough, UK) in continuous light (both tungsten and fluorescent lamps) at a photon fluence rate of 400 ~tmol-m -2.s-~ at 20~ and 80% relative humidity. Using an adaptation of the technique described by Kolderup (1979), radioactively labelled sucrose (2960 kBq [U-X4C] sucrose in 20 mM sucrose) was supplied to the plant. The labelled sucrose was placed in a reservoir attached to the stem and a small nick was made in the stem to allow uptake of the solution from the reservoir (see Fig. 1). Dark-treatments were provided by covering individual spikelets on one side of the ear with silver foil. Bright light was supplied to spikelets on the other side of the ear which were covered with clear polythene to mimic the humidity conditions of the dark-treated spikelets. Grains were taken from the basal florets of the spikelets in the central region of the spike at 0, 3, 5.5, 8.5, 11 and 14h after nicking the stem. The endosperm tissue was quickly excised and treated as described below in "analysis of radioactivity". In one experiment' plants were grown with ears covered with either cellophane bags or the same bags covered with black paper

Fig. 1. In-vivo method of supplying [14C]sucrose to wheat plants (adapted from Kolderup, 1979). Radioactively labelled sucrose (2960-kBq [U-14C]sucrose in 20 mM sucrose) was placed in a reservoir attached to the stem. Using the point of a hypodermic syringe (passed through the side-arm of the glass reservoir), a small nick was made in the stem to allow uptake of the solution from the reservoir

105 to exclude light. This treatment was imposed at 14 dpa to exclude effects on the cell-division phase of grain development. At grain maturity (55 dpa) grains from the central region of the spike were dried and weighed.

In-vitro incubation conditions. At 21 dpa, pairs of grain were removed from the basal florets of the spikelets in the central region of the spike and weighed. Grains were then bisected longitudinally along the crease region and were quickly transferred to 25-ml conical respirometer flasks with 3 ml of incubation medium that contained 10raM 2-(N-morpholino)ethanesulphonic acid (Mes) pH 6.5, 60 mM KC1 and a range of sucrose concentrations varying from 10 mM to 100 mM plus 37 kBq [14C]sucrose unless stated otherwise. In most experiments, the flasks were illuminated with two 300-W tungsten-halide lamps giving a photon fluence rate of 500 ~tmol.m 2.s-1 (Crump quantum photometer). A 3-cm-deep tray of water was placed between the light source and the flasks to prevent overheating. Different irradiances were obtained by varying the distance between the lamps and the flasks. Dark treatments were provided by covering the flasks with black tape. At the end of the incubation period the half-grains were removed from the incubation medium and briefly rinsed three times with 15 ml distilled water. The endosperm tissue was quickly excised and treated as described below in "analysis of radioactivity". Analysis of radioactivity from in-vivo and in-vitro treatments. Endosperm tissue from grain incubated in vivo or in vitro was homogenised in 2 ml of ice-cold 1 M perchloric acid (PCA) using a Polytron vortex homogeniser. The homogenisation probe was then washed with a further 1 ml of 1 M PCA, which was combined with the original PCA-homogenate. The tissue homogenates were centrifuged at 4000.g for 10 min at 4 ~ C, and the supernatant (PCAsoluble fraction) was decanted. The starchy pellet (PCA-insoluble fraction) was washed three times by resuspending in 3 ml of icecold distilled water and centrifuging as above. The supernatant of the first wash was combined with the PCA-soluble fraction and subsequent washes discarded. The PCA-soluble fraction was adjusted to pH 6.5-7.5 using 5 M K O H and 1 M KH2PO4. The insoluble potassium-perchlorate precipitate was removed by centrifugation at 4000-g for 10 min at 4 ~ C. The PCA-soluble fractions were kept at 4 ~ C during processing. The washed PCA-insoluble fraction was resuspended in 5 ml of 0.1 M sodium-acetate buffer pH 4.5, and boiled for 30 min with periodic shaking to resuspend the gelatinised starch. After cooling, the gelatinised starch was degraded enzymatically to glucose by incubation overnight at 37~ with 56 units of amyloglucosidase (EC 3.2.1.3; Sigma grade V). Any insoluble material remaining was removed by centrifugation at 4000.g for 15 min. Glucose in the supernatant fraction was measured using a Boehringer Kit (number 124010; Boehringer, Mannheim, FRG) which is based on the glucose-oxidase method described by Werner et al. (1970). Radioactivity in the supernatant of the enzyme-digested PCA-insoluble fraction, and in the PCA-soluble fraction was measured by liquid scintillation counting, after mixing 1 ml of the samples with 10 ml of lumagel scintillant (LKB Wallace Inc, Hinton, USA). Counting efficiency was determined in each sample by the sample channels ratio method. Measurement of grain oxygen exchange. At 21 dpa, pairs of grain were removed from the basal florets of the spikelets in the central region of the spike. Ten half-grains were prepared and incubated as described above under 'in-vitro incubation conditions', in 3 ml of incubation medium containing 100 mM sucrose. At various times during the incubation, the half-grains were briefly transferred to fresh medium in a Rank oxygen electrode (Rank Instruments, Bottisham, Cambridge, UK) to measure their oxygen consumption in darkness and oxygen evolution in bright light (500 lamol-m 2. S 1).

106

G.A. Foxon et al. : Starch synthesis in developing wheat grain

Results

The accumulation in vivo of radioactivity into the PCAsoluble fraction of endosperm tissue supplied with [~4C]sucrose proceeded at a steady rate over a 14-h period (Fig. 2a). The accumulation of radioactivity into the PCA-insoluble starch fraction showed an initial lag period of about 3 h before reaching a steady rate (Fig. 2 b). There was no effect of the light or dark treatments on the incorporation of radioactivity into the PCA-soluble or PCA-insoluble starch fractions (Fig. 2a, b). Similarly, there was no difference in grain dry weight at 55 dpa when ears were grown from 14 dpa in darkness or bright light (Table 1). When half-grains were incubated in vitro the accumulation of radioactivity from [~4C]sucrose into the PCA-insoluble starch fraction of endosperm tissue was stimulated about fivefold by incubating the grain in bright-light conditions (Fig. 3). This light-dependent increase in incorporation of [ 1 4 C ] s u c r o s e into starch reached a maximum at irradiances in excess of 200 gmol. m - 2 .s- ~. Incorporation of radioactivity from [14C]sucrose into the PCA-soluble fraction was unaffected between irradiances of 80 to 800 gmol.m-2, s-1, but was higher when grains were incubated in the dark (Fig. 3). The stimulation by light of [~*C]sucrose incorporation into starch in vitro was abolished when halfgrains were incubated in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) (Table 2). However, DCMU did not affect the accumulation of radioactivity into the PCA-soluble fraction (Table 2). The incorporation of [ 1 4 C ] s u c r o s e into the PCA-insoluble starch fraction was stimulated about threefold when half-grains were incubated in darkness under an atmosphere of oxygen instead of air (Table 3). No further increase was observed when the grains were incubated in bright light (Table 3). The oxygen treatment had no effect on the accumulation of radioactivity into the PCA-soluble fraction (Table 3). There was a steady increase in the amount of oxygen consumed by half-grains incubated in vitro (Fig. 4). In bright-light conditions the half-grains produced more oxygen by photosynthesis than was consumed by respi-

1000

8

1200. I000

&

600

800.

400

600

200 00 2, 4...... 6 8

400-

10

Time (hours)

12

14

Grain DW (mg.grain-1)

Uncovered, light Cellophane-covered, light Cellophane-covered, darkness

45 _+4 44_+ 5 42 4-_4

400

a ~o o

300

g~

200

L

PCA-soluble fraction

PCA-insoluble starch

o trt~

IO0

0

200

460

660

860

Photon fluence rate (.mol.m-2.s ')

Fig. 3. Effect of different irradiances on accumulation of radioactivity from sucrose into the PCA-soluble ( e - - e ) or PCA-insoluble ( z x zx) fraction of wheat endosperm incubated in vitro. The resuits are expressed as the mean + S E of five replicates each of two endosperms and errors were always less than 10% of the mean where not shown Table 2. The effect of 50 gm D C M U on the accumulation of radioactivity in the PCA-soluble or PCA-insoluble fractions of endosperm of wheat half-grains. The tissue was incubated in 3 ml of 100 m M sucrose and 37 kBq of [U-14C]sucrose at 25 ~ C. Light was supplied from two 300-W tungsten-halide lamps giving a photon fluence rate of 500 gmol- m 2. s - 1. Data are mean 4- SE (n = 5) Treatment

Control DCMU(50gM)

Radioactivity (Bq. endosperm- t) PCA-soluble

PCA-insoluble (starch)

Light

Light

Darkness

90.6+ 8.1 99.3+14.1 20.5+6.7 87.1___10.1 95.0+ 6.8 7.3+1.3

Darkness 9.1+2.2 8.0-t-2.2

in the PCA-soluble and PCA-insoluble fractions of wheat endosperm. The split grains were incubated in 3 ml of 50 m M sucrose with 333 kBq [U-t4C]sucrose at 15~ C. Oxygen was supplied by bubbling 100% 02 through the incubation media before starting the incubation and purging the flask with 02 during the incubation. Light was supplied as described in Table 2. Data are mean + SE (n = 5)

1400

800

Treatment

Table 3. The effect of oxygen on the accumulation of radioactivity

1600

o

Table 1. The effect of light or darkness on the dry weight of wheat grain. Separate spikes (ears only) of wheat were either left untreated or covered either in clear cellophane or clear cellophane covered with black paper at 14 dpa. At 55 dpa grains were harvested, dried and weighed. Data are mean -+ SE (n = 5)

200O~ ........ 0 2 4

Treatment 6

8

10

12

14

PCA-soluble

Time (hours)

Fig. 2a, b. Effect of light ( o - - - o ) or darkness ( A - - A ) on the accumulation of radioactivity from sucrose into the PCA-soluble fraction (a) or PCA-insoluble fraction (b) of endosperm of wheat grain under in-vivo conditions. The results are expressed as the mean and SE of seven replicates each of two endosperms

Radioactivity (Bq-endosperm- 1)

Aerated Oxygenated

PCA-insoluble

Light

Darkness

Light

Darkness

68.4_+7.7 75.5+7.8

73.3-I-5.1 73.5+7.8

30.0+3.0 33.2_+0.6

9.9_+2.4 31.4+2.7

G.A. Foxon et al. : Starch synthesis in developing wheat grain

Production

Net O2 balance (light-dark) 4

Oxygen

E

6

0

Consumption

1012-

14-

Time(hours)

Fig. 4. Changes in dark oxygen consumption ( A - - A ) and lightdependent oxygen production (o o) over a 5.5-h incubation period in vitro. Wheat half-grains were incubated as described in Material and methods and briefly transferred to a Rank oxygen electrode for measurement of oxygen exchange. Results are expressed as the mean of 10 grains

ration in the dark. However, because there was a steady rise in oxygen consumption by the grain and apparently no similar rise in photosynthesis, there was a gradual decline in net oxygen production by the grain. Thus, after 5 h of incubation in vitro, the grain consumed more oxygen by respiration than was produced by photosynthesis (Fig. 4). Discussion

The in-vivo experiments presented in this paper undermine the hypothesis that starch deposition in wheat endosperm is limited by oxygen derived from pericarp green-layer photosynthesis. When individual spikelets on an intact plant were grown in darkness or in bright-light conditions there was no detectable effect on the incorporation of [14C]sucrose into endosperm starch. These data are consistent with the finding that shading the developing spike (rather than shading the whole plant) has little influence on final grain yield (Thorne 1966). This information is also consistent with the observation that grain yield is not significantly depressed in the barley mutant Albino Lemma, which has a pericarp lacking chlorophyll (Duffus etal. 1985). Furthermore, when plants were grown to maturity with ears in either darkness or bright-light conditions there was no significant difference in dry weight between the light or dark treatments. Thus in vivo we were unable to detect any effect of light on endosperm starch synthesis or grain growth. In experiments where whole plants were shaded (Mengel and Judel 1981), there was a 20% decrease in grain yield. This effect may be attributed to a decrease in the supply of sucrose to the grain or to an interaction with phytohormones (Mengel and Judel 1981 ; Mengel et al. 1985), rather than to differences in the production of oxygen by photosynthesis in the pericarp.

107

Light does affect the incorporation of [14C]sucrose into starch under the in-vitro incubation conditions used here. The response to light of incubated half-grains was saturated by high photon fluence rates (in excess of 200 gmol. m-2. S-1), inhibited by the photosynthesis inhibitor DCMU, and could be mimicked by supplying supplementary oxygen. These findings substantiate those of Gifford and Bremner (1981) and we concur with their conclusion that the in-vitro "light-effect" is attributable to the photosynthetic production of oxygen by the pericarp green layer. This suggestion is itself consistent with the observation that oxygen exchange by cereal grain incubated in the light favours oxygen evolution rather than oxygen consumption (Nutbeam and Duffus 1978). We have made similar observations for half-grain incubated in vitro in conditions of high irradiance, but have extended the findings to show that half-grain oxygen consumption rises over threefold during a 5-h timecourse. Thus, net oxygen evolution gradually declined over this time-scale, so that by 5-6 h of incubation the rate of dark oxygen consumption exceeded the rate of photosynthetic oxygen production. It seems likely that the light effect can, at least in part, be attributed to the rising oxygen consumption of the grain cultured in vitro. As a result of this, some degree of endosperm anoxia is then inevitable, resulting in a decreased ability to synthesize starch. Nutbeam and Duffus (1978) speculated that oxygen derived from green-layer photosynthesis in cereal grain in vivo may limit endosperm starch synthesis. However, we have shown that starch synthesis is not stimulated in vivo by light. We conclude that either photosynthetic oxygen production by the pericarp may be limited in vivo by the shading caused by the bracts, palea and lemma surrounding the grain, or that atmospheric oxygen levels may be sufficient in vivo to support normal rates of starch synthesis. The elegant hypothesis (Gifford and Bremner 1981) that the light-oxygen effect provides an explanation for the shell structure of wheat starch granules (Buttrose 1965) now seems unlikely. Similarly, the proposals (Duffus 1979; Cochrane and Duffus 1979) that a major function of the green layer may be the provision of oxygen to maintain starch synthesis in endosperm tissue also seem untenable. An alternative role of the green layer may be to refix CO2 respired by the developing grain (Kriedemann 1966). The presence of phosphoenolpyruvate carboxylase and other enzymes of C4 photosynthesis in the pericarp tissues of barley (Duffus and Rosie 1973) and wheat grain (Wirth et al. 1977) together with the more direct evidence for C4 photosynthesis in barley grain (Nutbeam and Duffus 1976) are consistent with this proposal. Thus the presence of the green layer of the pericarp may represent an adaptation of this tissue for maintaining the efficiency of carbon deposition in the grain.

We thank Mrs. A.J. Foxon and Mr. P. James for their excellent technical assistance.

108

References Buttrose, M.S. (1962) The influence of environment on the shell structure of starch granules. J. Cell. Biol. 14, 159-167 Cochrane, M.P., Duffus, C.M. (1979) Morphology and ultrastructure of immature cereal grains in relation to transport. Ann. Bot. 44, 67-72 Duffus, C.M. (1979) Starch synthesis and grain growth. In: Crop physiology and cereal breeding, (Proc. Eucarpia Workshop, Wageningen, 1978) pp. 45-49, Spiertz, J.H.J., Kramer, Th., eds., UNIPUB, Vienna Duffus, C.M., Rosie, R. (1973) Some enzyme activities associated with the chlorophyll containing layers of the immature barley pericarp. Planta 114, 219-226 Duffus, C.M., Nutbeam, A.R., Scragg, P.A. (1985) Photosynthesis in the immature cereal pericarp in relation to grain growth. In: Regulation of sources and sinks in crop plants (Proc. British Plant Growth Regulator Group Symp. Monograph 12), pp. 243-256, Jeffcoat, B., Hawkins, A.F., Stead, A.D., eds. British Plant Growth Regulator Group Gifford, R.M., Bremner, P.M. (1981) Accumulation and conversion of sugars by developing wheat grains. II. Light requirement for kernels cultured in vitro. Aust. J. Plant Physiol. 8, 631-640 Kolderup, F. (1979) Interconversion of amino acids in maturing wheat grains. In: Seed protein improvements in cereals and grain legumes, vol. 1 (Proc. Int. Symp. Seed Protein Improvement in Cereals and Grain Legumes, 1978, Neuherberg, Germany), pp. 187-202, UNIPUB, Vienna

G.A. Foxon et al. : Starch synthesis in developing wheat grain Kriedemann, P. (1966) The photosynthetic activity of the wheat ear. Ann. Bot. 30, 349-363 Mengel, K., Judel, G.K. (1981) Effect of light intensity on the activity of starch synthesising enzymes and starch synthesis in developing wheat grains. Physiol. Plant. 51, 13-18 Mengel, K., Friedrich, B., Judel, G.K. (1985) Effect of light intensity on the concentrations of phytohormones in developing wheat grains. J. Plant Physiol. 120, 255 266 Nutbeam, A.R., Duffus, C.M. (1976) Evidence for C4 photosynthesis in barley pericarp tissue. Biochem. Biophys. Res. Comm. 70, 1198-1203 Nutbeam, A.R., Duffus, C.M. (1978) Oxygen exchange in the pericarp green layer of immature cereal grains. Plant Physiol. 60, 360-362 Radley, M. (1976) The development of the wheat grain in relation to endogenous growth substances. J. Exp. Bot. 27, 1009-1021 Thorne, G.N. (1966) Physiological aspects of grain yield in cereals. In: The growth of cereals and grasses, pp. 88-105, Millthorpe, F.L., Ivins, J.D., eds. Butterworths, London Werner, W., Rey, H.-G., Wielinger, H. (1970) fdber die Eigenschaften eines neuen Chromogens fiir die Blutzuckerbestimmung nach der GOD/POD-Methode. Z. Anal. Chem. 252, 224-228 Wirth, E., Kelly, G.J., Fischbeck, G., Latzko, E. (1977) Enzyme activities and products of CO2 fixation in various photosynthetic organs of wheat and oat. Z. Pflanzenphysiol. 82, 78-87

Received 10 August; accepted 1 November 1989