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Effect of grain location on the panicle on activities involved in starch synthesis in rice endosperm
Pergamon
0031-9422(94)E0109d
PhyturhPmurry. Vol. 36. No 4, pp. 843-847. 1994 Copyright $-I 1994 Elscv~er Sacna Ltd Pnnted I” Great Ehitam All rights rserved 0031-9422194 17.00+0.00
EFFECT OF GRAIN LOCATION ON THE PANICLE ON ACTIVITIES INVOLVED IN STARCH SYNTHESIS IN RICE ENDOSPERM TAKAYUKI UMEMOTO, YASUNORI NAKAMURA* and NORIMITSUISHIKURA Tohoku National Agricultural Experiment Station, Omagari, Akita 014-01, Japan; *National Institute of Agrobiological Resources, Tsukuba, lbaraki 305, Japan (Received
in
revised firm
Key Word ladex-Oryza satiua; rice; endosperm; caryopsis; amylose; starch.
13 January
biosynthesis;
1994)
granule-bound
starch synthase;
inferior
Abstract-Starch metabolism in rice endosperm of both the superior and inferior caryopses in the panicle was characterized by comparing changes in activities of five major enzymes associated with starch synthesis during endosperm development; sucrose synthase, ADPglucose pyrophosphorylase, Q-enzyme (starch branching enzyme), and soluble and starch granule-bound forms of starch synthase. Activities of ADPglucose pyrophosphorylase of both the superior and inferior caryopses increased markedly between 11 and 14 days after pollination. The developmental patterns of Q-enzyme activities were quite different between the superior and inferior caryopses. In addition, it was found that eight-14 days after pollination the activity of the starch granule-bound starch synthase in the inferior caryopsis was markedly lower than that in the superior caryopsis. These results suggest that the low activity of the granule-bound starch synthase is related to the lower content of amylose in the inferior caryopsis.
INTRODUCI’IOIV
Starch is the major component of the rice grain. Biosynthesis of starch in the endosperm is closely related to both yield and quality of rice. A rice panicle is composed of a number of caryopses. The grain of the superior caryopsis exhibits a faster rate of increase in dry weight during development, as well as a higher final dry weight than that of the inferior caryopsis [l-3]. Iwasaki et al. [3] reported on the process of nitrogen accumulation in the superior and inferior spikelets of rice. ‘5N-labelled nitrogen administered to rice plants at the early stages of ripening of grain was mainly incorporated into the superior spikelet, whereas the inferior spikelet could incorporate nitrogen during the late stages of ripening. Cellular structure also varies between the superior and inferior caryopses. Shimotsubo and Nakayama [4] reported that endosperm cells of the superior caryopsis are well-developed and regularly arranged from the central part toward the aleurone layer at day 5 after anthesis, but that endosperm development is delayed in the inferior caryopsis. The quality of starch in the rice grain differs depending on location, the endosperm of the superior caryopsis containing much more amylose than that of inferior grain [5]. There have been only a few reports regarding the activities of the enzymes responsible for starch biosynthesis in rice endosperm. Although developmental patterns of the enzymes involved in starch metabolism in rice endosperm have been reported by Baun et al. [6], and Nakamura and Yuki [7], the enzyme activities in both the superior and inferior caryopses were not compared. In
this investigation, we compared the activities of the enzymes involved in starch metabolism: sucrose synthase, ADPglucose pyrophosphorylase (ADPGlc PPase), Q-enzyme, a soluble form of starch synthase and a starch granule-bound form of starch synthase in the endosperm of the superior and inferior caryopses in the japonica rice cultivar 0~~316 during endosperm development. In addition, we measured the amylose content in both superior and inferior caryopses. RESULTS AND DlSCUS!!XON
The fresh weight of rice grains of both the superior and inferior caryopses increased greatly up to 14 days after pollination, while the dry weight further increased by ca two-fold thereafter (Table 1). Although the dry weight of the grain of the inferior caryopsis was only 20% of that of the superior caryopsis at five days after pollination, the former accounted for ca 70% of the latter 14 days after pollination. When the endosperm was fully mature, the fresh weight, as well as the dry weight of the inferior grains were ca 90% of those of the superior grains. The percent of starch in the inferior grains was the same as that of the superior grains (ca 60% of the dry weight of the grain) at the stage of maturation (26 and 31 days after pollination for superior and inferior caryopses, respectively) [Table 21. However, the amylose content in the grain of the inferior caryopsis was markedly lower than that of the superior caryopsis (s5% of that of the superior caryopsis), consistent with the results reported by Horiuchi [S]. 843
T. UMEMOTO et 01.
844
Table
and inferior
-
100
1. Changes in fresh and dry weight of grains of superior
(A)
rice caryopses
3
Days after pollination 5
8
11
14
26
SO
31
(mg grain _ ‘) Fresh weight Superior Inferior
grain grain
6.3
15.4
19.6
22.7
1.5
10.0
13.9
18.7
1.2
4.6
8.1
11.7
0.2
2.3
4.3
8.3
27.1 cl
24.3
“.
Superior Inferior
8
5
Dry weight grain grain
21.1
i- -
18.6
14
11
3oo r(B)
Values are the means of IO grains.
Activity patterns of the major enzymes involved in starch metabolism were examined during endosperm development of superior and inferior caryopses (Figs 1 and 2). In rice endosperm, sucrose synthase catalyses sucrose which is translocated from leaves [g]. The increase in the enzyme activity per endosperm was pronounced during the early stage of the development, up to eight days after pollination, in both the superior and inferior caryopses (Fig. IA). It is known that ADPGlc PPase is a regulatory enzyme catalysing the rate-limiting step of starch biosynthesis in both photosynthetic and nonphotosynthetic plant tissues [9, lo]. In contrast to sucrose synthase activity, increases in the activities of ADPGlc PPase of the endosperm of both the superior and inferior caryopses were comparatively slow up to 11 days after pollination, then the increases were markedly accelerated between I I and 14 days after pollination (Fig. 1B). Starch branching enzyme or Q-enzyme produces amylopectin, the major component of starch in plants, by introducing branches of cr-1,6-glycosidic linkages into a1,4-glucans. The patterns of Q-enzyme activity during endosperm development were quite different between the endosperm of the superior and inferior caryopses (Fig. 1C). The level of Q-enzyme activity in the endosperm of the inferior caryopsis accounted for only 17% of that of superior caryopsis at eight days after pollination, but the activity in the inferior caryopsis increased rapidly by ca six-fold eight-l 1 days after pollination. The Q-enzyme activity in the endosperm of the inferior caryopsis was 72% of that of the superior caryopsis at 11 days after
Table
2. Dry wt. starch content inferior
Superior Inferior
grain grain
”
5
5
8
11
14
8
11
14
Days after pollination Fig.
1. Changes in activities of sucrose synthase (A), ADPglu-
case pyrophosphorylase endosperm
of superior
(B) and Q-enzyme (C) in developing rice
(0)
and inferior
(0)
caryopses. Vertical
bars denote s.d. (n = 4, sucrose synthase and Q-enzyme; ADPglucose
n = 3.
pyrophosphorylase).
pollination. Starch synthase catalyses the irreversible reaction in which the a- 1,4-glycosidic linkage is elongated into r-glucans. The activity of a soluble form of starch synthase rose progressively with time up to 1 I days after pollination in the endosperm of the superior and inferior caryopses (Fig. 2). By contrast, the activity of a granulebound form of the enzyme was low up to eight days but
and amylose content of grains of superior and
rice caryopses at the mature stage
Dry weight
Starch content
Amylose content
(mg grain - i)
(% dry wt)
(% starch)
22.8
61.3k2.6
17.98 + 0.24
19.2
62.4 f 2.6
15.37+0.19
Values for starch and amylose content are the means +s.d. of three measurements, and those for dry wt are the means of 10 grains.
845
Starch synthesis in rice endosperm Table 3. Relative rates of enzyme activities in endosperm of inferior and superior rice caryopse-s Days after pollination
0’
5
8
11
14
26
32
Days after pollution Fig. 2. Changes in activities of soluble starch synthase and granule-bound starch synthase in developing rice endosperm of superior (0, soluble; A, granule-bound) and inferior (0. soluble; A, granule-bound) caryopses. Vertical bars denote s.d. (n = 3).
Enzymes
8
11
Sucrose synthase ADPGlc PPase Q-enzyme Starch synthase soluble granule-bound
57 60 17
(Per cent) 49 65 51 70 72 80
65 29
68 31
14
63 38
Table 4. Relative enzyme activities in endosperm of superior and inferior rice caryopses on a dry weight basis Days after pollination
then increased markedly up to 14 days after pollination, and the increase seemed to continue until the latest stage of grain development (Fig. 2). The ratios of the activities of the granule-bound starch synthase to the soluble starch synthase increased with the developmental stages in the endosperm of both the superior and inferior caryopses. This result is in agreement with the previous report by Nakamura and Yuki [7]. It has also been reported that amylose content increases with the progression of endosperm development in rice [ll]. The present data support the idea that the synthesis of amylose is mediated by the starch granule-bound starch synthase, but not by the soluble starch synthase. The developmental patterns of the enzymes in the inferior caryopsis were compared with those of the superior caryopsis (Tables 3 and 4). The ratios of enzyme activities on a per endosperm basis in the inferior caryopsis to those in the superior were between 50 and 80% with respect to sucrose synthase, ADPGlc PPase, the soluble starch synthase and Q-enzyme, except that the ratio of Q-enzyme at eight days after pollination was only 17%. By contrast, the ratios of granule-bound starch synthase were between 30 and 40% during days 8- 14 after pollination (Table 3). The similar trend was confirmed when the enzyme activities were compared on the basis of grain dry weight (Table 4). The bound starch synthase, sometimes called a Waxy protein, has been reported to be indispensable for amylose biosynthesis [12]. The present data also suggest that the lower activity of the granulebound starch synthase in the endosperm of the inferior caryopsis as compared to four other enzymes is related to the lower amount of amylose in the inferior grain compared with the superior grain.
EXPERIMENTAL
Plant material. Twenty seeds of japonica rice (Oryra sativa cv 0~~316) were directly seeded in a circle in
plastic pots and grown in a greenhouse without temp.
Enzymes
8
11 (Activity) 9.85 9.12
14
1.05 6.49
Sucrose synthase’
Superior Inferior
14.9 16.9
ADPGlc
Superior Inferior
(113) 15.5 18.5
(93) 13.7 13.3
(92) 20.2 19.8
Superior Inferior
(119) 2.87 0.95
(97) 2.36 3.19
(98) 1.65 1.87
(33)
(135)
(113)
PPase*
Q-enzyme?
Starch synthasc soluble*
granule-bound*
Superior Inferior
0.57 0.74
0.47 0.61
0.33 0.29
Superior Inferior
(130) 0.030 0.018
(130) 0.073 0.042
(88) 0.085 0.046
(60)
(58)
04)
Data were calculated from results shown in Table 1 and Figs 1 and 2. Numbers in parentheses indicate ratios of enzyme activities of the inferior caryopsis to the superior caryopsis. lnmol min-’ mgdry wt-‘. tunit min-’ mgdry wt-‘.
control under natural daylight from April to September in Omagari, Akita, Japan. Figure 3 indicates the location of the samples of the superior and inferior caryopses. The samples were marked at the day of pollination and grains collected at 5,8,11 and 14 days after pollination, as well as at the mature stage. Grains were stored at -85” until used for expts. The developmental stages of the superior caryopsis (days after pollination) were as follows: S{maximum grain length}; 8-{maximum grain width}; 11-{milk-ripe stage}; lC{dough-ripe stage}. The stages of the inferior caryopsis were delayed, the grains at 14 days after pollination corresponding to the milk-ripe stage. Fr. wts are the mean values of 10 grains. Dry wt was determined after drying the grains at 80” for 48 hr.
846
T. UMEMOTO et ul.
Superior caryopseli
Inferior caryopscs
Fig. 3. Diagram of caryopses on a rice panicle. Circles indicate the location of caryopses. Closed circles indicate caryopses used for the experiment.
Starch content. Starch content and amylose content were measured at the mature grain stage. To measure starch content, ca 5 g of brown rice grain was crushed with a grain crusher. The crushed sample was passed through a 50-mesh sieve. DMSO (20 ml) and 8 N HCl (5 ml) were added to the rice powder which was then incubated at 60” for 30 min. The starch soln was added to 50 ml of H,O, the pH adjusted to 4-5 with 5 N NaOH and the soln made up to lOOmI. This soln was then diluted 100-fold with Hz0 and 100~1 added to 200 ~1 HZ0 and incubated with 100 ~1 of 100 mM Na-acetate buffer (pH 4.8) containing amyloglucosidase (3 U) for 1 hr at 55”. The reaction was terminated by heating the mixt. at 100” for 1 min. The soln was transferred into an Eppendorf tube and centrifuged at I5 000 rpm for 10 min. A portion (3OOnl) of the supernatant was taken and mixed with 200~1 of 150 mM Hepes-NaOH buffer (pH 7.4) containing IO mM MgSO,, 3.2 mM NADP and 10.8 mM ATP. Starch content was assayed by measuring the increase in A at 340 nm after the addition of I ~1 each of hexokinase (1.4 U) and glucose-6-phosphate dehydrogenase (0.35 U). Amyiose mnfmt. Prepn of starch was conducted essentially by the method of ref. [13]. i.e., ca 5 g of brown rice grain was milled to give co 90% (w/w) milled rice. The rice powder was suspended in ca 75 ml of chilled 0.2% NaOH and stirred for 3 hr at 5”. After washing with Hz0 until it became neutral, the starch sample was passed through a 200-mesh sieve and dried under red. pres. at 30”. Debranthing of starch with isoamylase and fractionation of debranched starch on Sephadex G-75 column was performed according to the method of ref. [14]. Starches (cu 40 mg) were gelatinized with 1 N NaOH overnight at 5”. Gelatinized starches were neutralized with 1 N HCl and debranched by crystalline Pseudomonas isoamylase (1.475 U) in 30 mM Na-acetate buffer, pH 3.5, at 40” for 24 hr. Debranched starches were dried under red. pres. at 40” and dissolved in 1 N NaOH. The soln (2 ml) was applied to a Sephadex G-75 column (22 mm diameter x95 cm length), which had been equilibrated with
0.02 N NaOH containing 0.2% NaCl. The starch sample was eluted with the same soln at a flow rate of 0.25 ml min- l. Frs were collected at 5 ml intervals and each fr. was neutralized with 1 N HCI. The carbohydrate content in each fr. was measured by the phenol- HzSOh method [lS]. Amylose and amylopectin were divided according to the following E.,,, of A of I,-carbohydrate complexes in each tube, amylose; &,,, 26620 nm, amylopectin: L,,, < 620 nm [ t 63. Enzyme preparation. All procedures were performed at O-4,‘. Caryopses were dehulled and lOdehulled grains were used for expts. The embryo and pericarp were removed from dehulled grain and the endosperm was ground in a chilled glass homogenizer with 2 ml of 1OOmM Tricine-NaOH buffer (pH 8) containing 8 mM MgCI,, 2 mM EDTA, 50 mM 2-mercaptoethanol and 12.5% (v/v) glycerol. Since it is difficult to remove the peticarp from the grain at the earlier stages ofendosperm development, the endosperm was pushed out into the buffer with a glass rod and homogenized in a glass homogenizer. The homogenate was transferred to an Eppendorf tube and centrifuged at 15000 rpm for 5 min. The supernatant was used for assays of sucrose synthase, ADPGlc PPase, Q-enzyme and soluble starch synthase. The ppt. was washed twice with 1 ml of a grinding soln and suspended in t ml of the same sofn for the assay of granule-bound starch synthase. Enzyme assays. Enzyme activities were measured according to the method described in ref. [8] for ADPGlc PPase, Q-enzyme, and the soluble and granule-bound starch synthase, and that of ref. [17] for sucrose synthase. All enzyme assays were performed in a range where the velocity was proportional to enzyme concn and incubation time. Each result is the mean ~s.d. of at least three replicate incubations. Ackno~~ed~eme~ts --We thank MS Tetsu Toshima and
MS Kimiko Ito for their technical assistance. This work was supported by a grant for the encouragement of basic research at National Research Institutes from the Science and Technology Agency of Japan, and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan. REFERENCES
I. Nagato, K. (1941) Proc, Crop Sci. Sot. Jpn 13, 156. 2. Arai, K. and Kono, Y. (1979) Jpn J. Crop Sci. 48,335. 3. Iwasaki, Y., Mae, T., Makino, A., Ohira, K. and Ojima, K. (1992) Soil Sci. Phnt Nutr. 38, 517. 4. Shimotsubo, K. and Nakayama, H. (1974) Bull. Nokuriku Nutl. Ayric. Erp. Sta. 16, 29.
5. Horiuchi, H. (1991) Jpn J. Breed. 41 (Suppl. 2), 544. 6. Baun, L. C., Palmino, E. P., Perez, C. M. and Juliano, B. 0. (1970) Phr PhJsiol. 46, 429. 7. Nakamura, Y. and Yuki, K. (1992) PIant Sci. 82, 15. 8. Nakamura, Y., Yuki, K., Park, S. and Ohya, T. (1989) Phf Cell Physiol. 30, 833. 9. Preiss, J. and Levi, C. (1980) in The Biochemistry oj Plants (Preiss, J., ed.), Vol. 3. p. 37 1. Academic Press, New York.
Starch synthesis in rice endospem 10. Stark, D. M., Timmerman, K. P., Barry, G. F., Preiss, J. and Kishore, G. M. (1992) Science 258, 287. 11. Sano, Y. (1984) Theor. Appl. Genet. 68, 467. 12. Asaoka, M., Okuno, K., Sugimoto, Y. and Fuwa, H. (1985) Agric. Biol. Chem. 49, 1973. 13. Yamamoto, K., Sawada, S. and Onogaki, I. (1973) Denpun 14.
Kagaku
20,W.
Ikawa, Y., Glover, D. V., Sugimoto, Y. and Fuwa, H. (1981) Starch 33, 9.
a41
15. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F. (1956) Analyt. Chem. 28. 350. 16. Sugimoto, Y., Yamada, K., Sakamoto, S. and Fuwa, H. (1981) Starch 33, 112. 17. Kerr, P. S., Huber, S. C. and Israel, D. W. (1984) Plant Physiol. 75, 883.
0031-9422(94)E0109d
PhyturhPmurry. Vol. 36. No 4, pp. 843-847. 1994 Copyright $-I 1994 Elscv~er Sacna Ltd Pnnted I” Great Ehitam All rights rserved 0031-9422194 17.00+0.00
EFFECT OF GRAIN LOCATION ON THE PANICLE ON ACTIVITIES INVOLVED IN STARCH SYNTHESIS IN RICE ENDOSPERM TAKAYUKI UMEMOTO, YASUNORI NAKAMURA* and NORIMITSUISHIKURA Tohoku National Agricultural Experiment Station, Omagari, Akita 014-01, Japan; *National Institute of Agrobiological Resources, Tsukuba, lbaraki 305, Japan (Received
in
revised firm
Key Word ladex-Oryza satiua; rice; endosperm; caryopsis; amylose; starch.
13 January
biosynthesis;
1994)
granule-bound
starch synthase;
inferior
Abstract-Starch metabolism in rice endosperm of both the superior and inferior caryopses in the panicle was characterized by comparing changes in activities of five major enzymes associated with starch synthesis during endosperm development; sucrose synthase, ADPglucose pyrophosphorylase, Q-enzyme (starch branching enzyme), and soluble and starch granule-bound forms of starch synthase. Activities of ADPglucose pyrophosphorylase of both the superior and inferior caryopses increased markedly between 11 and 14 days after pollination. The developmental patterns of Q-enzyme activities were quite different between the superior and inferior caryopses. In addition, it was found that eight-14 days after pollination the activity of the starch granule-bound starch synthase in the inferior caryopsis was markedly lower than that in the superior caryopsis. These results suggest that the low activity of the granule-bound starch synthase is related to the lower content of amylose in the inferior caryopsis.
INTRODUCI’IOIV
Starch is the major component of the rice grain. Biosynthesis of starch in the endosperm is closely related to both yield and quality of rice. A rice panicle is composed of a number of caryopses. The grain of the superior caryopsis exhibits a faster rate of increase in dry weight during development, as well as a higher final dry weight than that of the inferior caryopsis [l-3]. Iwasaki et al. [3] reported on the process of nitrogen accumulation in the superior and inferior spikelets of rice. ‘5N-labelled nitrogen administered to rice plants at the early stages of ripening of grain was mainly incorporated into the superior spikelet, whereas the inferior spikelet could incorporate nitrogen during the late stages of ripening. Cellular structure also varies between the superior and inferior caryopses. Shimotsubo and Nakayama [4] reported that endosperm cells of the superior caryopsis are well-developed and regularly arranged from the central part toward the aleurone layer at day 5 after anthesis, but that endosperm development is delayed in the inferior caryopsis. The quality of starch in the rice grain differs depending on location, the endosperm of the superior caryopsis containing much more amylose than that of inferior grain [5]. There have been only a few reports regarding the activities of the enzymes responsible for starch biosynthesis in rice endosperm. Although developmental patterns of the enzymes involved in starch metabolism in rice endosperm have been reported by Baun et al. [6], and Nakamura and Yuki [7], the enzyme activities in both the superior and inferior caryopses were not compared. In
this investigation, we compared the activities of the enzymes involved in starch metabolism: sucrose synthase, ADPglucose pyrophosphorylase (ADPGlc PPase), Q-enzyme, a soluble form of starch synthase and a starch granule-bound form of starch synthase in the endosperm of the superior and inferior caryopses in the japonica rice cultivar 0~~316 during endosperm development. In addition, we measured the amylose content in both superior and inferior caryopses. RESULTS AND DlSCUS!!XON
The fresh weight of rice grains of both the superior and inferior caryopses increased greatly up to 14 days after pollination, while the dry weight further increased by ca two-fold thereafter (Table 1). Although the dry weight of the grain of the inferior caryopsis was only 20% of that of the superior caryopsis at five days after pollination, the former accounted for ca 70% of the latter 14 days after pollination. When the endosperm was fully mature, the fresh weight, as well as the dry weight of the inferior grains were ca 90% of those of the superior grains. The percent of starch in the inferior grains was the same as that of the superior grains (ca 60% of the dry weight of the grain) at the stage of maturation (26 and 31 days after pollination for superior and inferior caryopses, respectively) [Table 21. However, the amylose content in the grain of the inferior caryopsis was markedly lower than that of the superior caryopsis (s5% of that of the superior caryopsis), consistent with the results reported by Horiuchi [S]. 843
T. UMEMOTO et 01.
844
Table
and inferior
-
100
1. Changes in fresh and dry weight of grains of superior
(A)
rice caryopses
3
Days after pollination 5
8
11
14
26
SO
31
(mg grain _ ‘) Fresh weight Superior Inferior
grain grain
6.3
15.4
19.6
22.7
1.5
10.0
13.9
18.7
1.2
4.6
8.1
11.7
0.2
2.3
4.3
8.3
27.1 cl
24.3
“.
Superior Inferior
8
5
Dry weight grain grain
21.1
i- -
18.6
14
11
3oo r(B)
Values are the means of IO grains.
Activity patterns of the major enzymes involved in starch metabolism were examined during endosperm development of superior and inferior caryopses (Figs 1 and 2). In rice endosperm, sucrose synthase catalyses sucrose which is translocated from leaves [g]. The increase in the enzyme activity per endosperm was pronounced during the early stage of the development, up to eight days after pollination, in both the superior and inferior caryopses (Fig. IA). It is known that ADPGlc PPase is a regulatory enzyme catalysing the rate-limiting step of starch biosynthesis in both photosynthetic and nonphotosynthetic plant tissues [9, lo]. In contrast to sucrose synthase activity, increases in the activities of ADPGlc PPase of the endosperm of both the superior and inferior caryopses were comparatively slow up to 11 days after pollination, then the increases were markedly accelerated between I I and 14 days after pollination (Fig. 1B). Starch branching enzyme or Q-enzyme produces amylopectin, the major component of starch in plants, by introducing branches of cr-1,6-glycosidic linkages into a1,4-glucans. The patterns of Q-enzyme activity during endosperm development were quite different between the endosperm of the superior and inferior caryopses (Fig. 1C). The level of Q-enzyme activity in the endosperm of the inferior caryopsis accounted for only 17% of that of superior caryopsis at eight days after pollination, but the activity in the inferior caryopsis increased rapidly by ca six-fold eight-l 1 days after pollination. The Q-enzyme activity in the endosperm of the inferior caryopsis was 72% of that of the superior caryopsis at 11 days after
Table
2. Dry wt. starch content inferior
Superior Inferior
grain grain
”
5
5
8
11
14
8
11
14
Days after pollination Fig.
1. Changes in activities of sucrose synthase (A), ADPglu-
case pyrophosphorylase endosperm
of superior
(B) and Q-enzyme (C) in developing rice
(0)
and inferior
(0)
caryopses. Vertical
bars denote s.d. (n = 4, sucrose synthase and Q-enzyme; ADPglucose
n = 3.
pyrophosphorylase).
pollination. Starch synthase catalyses the irreversible reaction in which the a- 1,4-glycosidic linkage is elongated into r-glucans. The activity of a soluble form of starch synthase rose progressively with time up to 1 I days after pollination in the endosperm of the superior and inferior caryopses (Fig. 2). By contrast, the activity of a granulebound form of the enzyme was low up to eight days but
and amylose content of grains of superior and
rice caryopses at the mature stage
Dry weight
Starch content
Amylose content
(mg grain - i)
(% dry wt)
(% starch)
22.8
61.3k2.6
17.98 + 0.24
19.2
62.4 f 2.6
15.37+0.19
Values for starch and amylose content are the means +s.d. of three measurements, and those for dry wt are the means of 10 grains.
845
Starch synthesis in rice endosperm Table 3. Relative rates of enzyme activities in endosperm of inferior and superior rice caryopse-s Days after pollination
0’
5
8
11
14
26
32
Days after pollution Fig. 2. Changes in activities of soluble starch synthase and granule-bound starch synthase in developing rice endosperm of superior (0, soluble; A, granule-bound) and inferior (0. soluble; A, granule-bound) caryopses. Vertical bars denote s.d. (n = 3).
Enzymes
8
11
Sucrose synthase ADPGlc PPase Q-enzyme Starch synthase soluble granule-bound
57 60 17
(Per cent) 49 65 51 70 72 80
65 29
68 31
14
63 38
Table 4. Relative enzyme activities in endosperm of superior and inferior rice caryopses on a dry weight basis Days after pollination
then increased markedly up to 14 days after pollination, and the increase seemed to continue until the latest stage of grain development (Fig. 2). The ratios of the activities of the granule-bound starch synthase to the soluble starch synthase increased with the developmental stages in the endosperm of both the superior and inferior caryopses. This result is in agreement with the previous report by Nakamura and Yuki [7]. It has also been reported that amylose content increases with the progression of endosperm development in rice [ll]. The present data support the idea that the synthesis of amylose is mediated by the starch granule-bound starch synthase, but not by the soluble starch synthase. The developmental patterns of the enzymes in the inferior caryopsis were compared with those of the superior caryopsis (Tables 3 and 4). The ratios of enzyme activities on a per endosperm basis in the inferior caryopsis to those in the superior were between 50 and 80% with respect to sucrose synthase, ADPGlc PPase, the soluble starch synthase and Q-enzyme, except that the ratio of Q-enzyme at eight days after pollination was only 17%. By contrast, the ratios of granule-bound starch synthase were between 30 and 40% during days 8- 14 after pollination (Table 3). The similar trend was confirmed when the enzyme activities were compared on the basis of grain dry weight (Table 4). The bound starch synthase, sometimes called a Waxy protein, has been reported to be indispensable for amylose biosynthesis [12]. The present data also suggest that the lower activity of the granulebound starch synthase in the endosperm of the inferior caryopsis as compared to four other enzymes is related to the lower amount of amylose in the inferior grain compared with the superior grain.
EXPERIMENTAL
Plant material. Twenty seeds of japonica rice (Oryra sativa cv 0~~316) were directly seeded in a circle in
plastic pots and grown in a greenhouse without temp.
Enzymes
8
11 (Activity) 9.85 9.12
14
1.05 6.49
Sucrose synthase’
Superior Inferior
14.9 16.9
ADPGlc
Superior Inferior
(113) 15.5 18.5
(93) 13.7 13.3
(92) 20.2 19.8
Superior Inferior
(119) 2.87 0.95
(97) 2.36 3.19
(98) 1.65 1.87
(33)
(135)
(113)
PPase*
Q-enzyme?
Starch synthasc soluble*
granule-bound*
Superior Inferior
0.57 0.74
0.47 0.61
0.33 0.29
Superior Inferior
(130) 0.030 0.018
(130) 0.073 0.042
(88) 0.085 0.046
(60)
(58)
04)
Data were calculated from results shown in Table 1 and Figs 1 and 2. Numbers in parentheses indicate ratios of enzyme activities of the inferior caryopsis to the superior caryopsis. lnmol min-’ mgdry wt-‘. tunit min-’ mgdry wt-‘.
control under natural daylight from April to September in Omagari, Akita, Japan. Figure 3 indicates the location of the samples of the superior and inferior caryopses. The samples were marked at the day of pollination and grains collected at 5,8,11 and 14 days after pollination, as well as at the mature stage. Grains were stored at -85” until used for expts. The developmental stages of the superior caryopsis (days after pollination) were as follows: S{maximum grain length}; 8-{maximum grain width}; 11-{milk-ripe stage}; lC{dough-ripe stage}. The stages of the inferior caryopsis were delayed, the grains at 14 days after pollination corresponding to the milk-ripe stage. Fr. wts are the mean values of 10 grains. Dry wt was determined after drying the grains at 80” for 48 hr.
846
T. UMEMOTO et ul.
Superior caryopseli
Inferior caryopscs
Fig. 3. Diagram of caryopses on a rice panicle. Circles indicate the location of caryopses. Closed circles indicate caryopses used for the experiment.
Starch content. Starch content and amylose content were measured at the mature grain stage. To measure starch content, ca 5 g of brown rice grain was crushed with a grain crusher. The crushed sample was passed through a 50-mesh sieve. DMSO (20 ml) and 8 N HCl (5 ml) were added to the rice powder which was then incubated at 60” for 30 min. The starch soln was added to 50 ml of H,O, the pH adjusted to 4-5 with 5 N NaOH and the soln made up to lOOmI. This soln was then diluted 100-fold with Hz0 and 100~1 added to 200 ~1 HZ0 and incubated with 100 ~1 of 100 mM Na-acetate buffer (pH 4.8) containing amyloglucosidase (3 U) for 1 hr at 55”. The reaction was terminated by heating the mixt. at 100” for 1 min. The soln was transferred into an Eppendorf tube and centrifuged at I5 000 rpm for 10 min. A portion (3OOnl) of the supernatant was taken and mixed with 200~1 of 150 mM Hepes-NaOH buffer (pH 7.4) containing IO mM MgSO,, 3.2 mM NADP and 10.8 mM ATP. Starch content was assayed by measuring the increase in A at 340 nm after the addition of I ~1 each of hexokinase (1.4 U) and glucose-6-phosphate dehydrogenase (0.35 U). Amyiose mnfmt. Prepn of starch was conducted essentially by the method of ref. [13]. i.e., ca 5 g of brown rice grain was milled to give co 90% (w/w) milled rice. The rice powder was suspended in ca 75 ml of chilled 0.2% NaOH and stirred for 3 hr at 5”. After washing with Hz0 until it became neutral, the starch sample was passed through a 200-mesh sieve and dried under red. pres. at 30”. Debranthing of starch with isoamylase and fractionation of debranched starch on Sephadex G-75 column was performed according to the method of ref. [14]. Starches (cu 40 mg) were gelatinized with 1 N NaOH overnight at 5”. Gelatinized starches were neutralized with 1 N HCl and debranched by crystalline Pseudomonas isoamylase (1.475 U) in 30 mM Na-acetate buffer, pH 3.5, at 40” for 24 hr. Debranched starches were dried under red. pres. at 40” and dissolved in 1 N NaOH. The soln (2 ml) was applied to a Sephadex G-75 column (22 mm diameter x95 cm length), which had been equilibrated with
0.02 N NaOH containing 0.2% NaCl. The starch sample was eluted with the same soln at a flow rate of 0.25 ml min- l. Frs were collected at 5 ml intervals and each fr. was neutralized with 1 N HCI. The carbohydrate content in each fr. was measured by the phenol- HzSOh method [lS]. Amylose and amylopectin were divided according to the following E.,,, of A of I,-carbohydrate complexes in each tube, amylose; &,,, 26620 nm, amylopectin: L,,, < 620 nm [ t 63. Enzyme preparation. All procedures were performed at O-4,‘. Caryopses were dehulled and lOdehulled grains were used for expts. The embryo and pericarp were removed from dehulled grain and the endosperm was ground in a chilled glass homogenizer with 2 ml of 1OOmM Tricine-NaOH buffer (pH 8) containing 8 mM MgCI,, 2 mM EDTA, 50 mM 2-mercaptoethanol and 12.5% (v/v) glycerol. Since it is difficult to remove the peticarp from the grain at the earlier stages ofendosperm development, the endosperm was pushed out into the buffer with a glass rod and homogenized in a glass homogenizer. The homogenate was transferred to an Eppendorf tube and centrifuged at 15000 rpm for 5 min. The supernatant was used for assays of sucrose synthase, ADPGlc PPase, Q-enzyme and soluble starch synthase. The ppt. was washed twice with 1 ml of a grinding soln and suspended in t ml of the same sofn for the assay of granule-bound starch synthase. Enzyme assays. Enzyme activities were measured according to the method described in ref. [8] for ADPGlc PPase, Q-enzyme, and the soluble and granule-bound starch synthase, and that of ref. [17] for sucrose synthase. All enzyme assays were performed in a range where the velocity was proportional to enzyme concn and incubation time. Each result is the mean ~s.d. of at least three replicate incubations. Ackno~~ed~eme~ts --We thank MS Tetsu Toshima and
MS Kimiko Ito for their technical assistance. This work was supported by a grant for the encouragement of basic research at National Research Institutes from the Science and Technology Agency of Japan, and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan. REFERENCES
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a41
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