Activities of enzymes involved in sucrose-to-starch metabolism in rice grains subjected to water stress during filling

Field Crops Research 81 (2003) 69±81

Activities of enzymes involved in sucrose-to-starch metabolism in rice grains subjected to water stress during ®lling Jianchang Yanga, Jianhua Zhangb,*, Zhiqin Wanga, Qingsen Zhua, Lijun Liua a

College of Agriculture, Yangzhou University, Yangzhou, Jiangsu, China Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China

b

Received 27 March 2002; received in revised form 12 September 2002; accepted 10 October 2002

Abstract Slow grain ®lling of rice (Oryza sativa L.) is a problem due to the heavy use of nitrogen fertilizer or the high lodging resistance of some cultivars. This study investigated if controlled water de®cit during grain ®lling could enhance sink strength by regulating key enzymes involved and lead to faster grain ®lling. Two rice cultivars with high lodging resistance and slow grain ®lling were grown in the ®eld and treated with either normal nitrogen (NN) or high nitrogen (HN) at heading. Well-watered (WW) and water-de®cit stressed (WS) treatments were imposed from 9 days after anthesis (DAA) until maturity. Leaf water potentials of both cultivars markedly decreased during the day as a result of WS treatments, but completely recovered by early morning. WS promoted the reallocation of pre®xed 14 C from stems to grains, facilitated starch accumulation in grains and increased grain®lling rate although it shortened grain-®lling period. In contrast, HN behaved in the opposite way. Sucrose synthase (EC 2.4.1.13) activity was substantially enhanced by water stress, and was positively correlated with starch accumulation rate (SAR) in the grains. Both soluble and insoluble invertase (EC 3.2.1.26) activities were less enhanced by WS and showed no signi®cant correlation with SAR. Starch branching enzyme (BE) (EC 2.4.1.18) and soluble starch synthase (EC 2.4.1.18) activities were also enhanced by the WS, with the former enhanced more than the latter, and were signi®cantly correlated with SAR. Adenine diphosphoglucose pyrophosphorylase (EC 2.7.7.27) activity was little affected by WS. The results suggest that WS-increased remobilization and grain-®lling rate were attributed to enhanced sink strength by regulating sucrose synthase and starch BE activities in rice grains when subjected to water stress during the grain-®lling period. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Rice (Oryza sativa L.); Water stress; Carbon metabolism; Sink strength; Sucrose synthase; Starch branching enzyme

1. Introduction Starch in rice (Oryza sativa L.) grains contributes about 90% of the ®nal dry weight of an unpolished grain (Yoshida, 1972; Cao et al., 1992). Grain ®lling is actually a process of starch accumulation. It has been reported that there are 33 major enzymes involved in * Corresponding author. Fax: ‡852-3411-7350. E-mail address: [email protected] (J. Zhang).

the metabolism of carbohydrates in developing rice endosperm (Nakamura et al., 1989). Among them, however, four enzymes are considered to play a key role in this process (Nakamura et al., 1989; Preiss et al., 1991; Kato, 1995; Yang et al., 2001a). They are: sucrose synthase (EC 2.4.1.13, SuSase), adenine diphosphoglucose pyrophosphorylase (EC 2.7.7.27, AGPase), starch synthase (EC 2.4.1.21, StSase), and starch branching enzyme (BE) (EC 2.4.1.18, BE). As sucrose is the main transported form of assimilates in

0378-4290/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 2 9 0 ( 0 2 ) 0 0 2 1 4 - 9

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rice plants, the ®rst step in the sucrose-to-starch pathway is to cleave sucrose to its constituent monosaccharides, which can then be used either in metabolic or biosynthetic reactions (Ranwala and Miller, 1998). In rice grains, SuSase acts as the ®rst step in the pathway (Kato, 1995), and its activity has accordingly been linked to sink strength in the developing grain (Liang et al., 2001). The predominant pathway for starch biosynthesis in plants is composed of three reactions catalyzed by AGPase, StSase, and BE (Nakamura et al., 1989; Preiss et al., 1991; Okita, 1992; Martin and Smith, 1995). The activities of the three enzymes are closely associated with the increase in starch content during development of rice endosperm (Nakamura and Yuki, 1992). Improving grain ®lling is vitally important in cases where slow grain ®lling is a problem due to the heavy use of nitrogen fertilizer or the high lodging-resistant feature of a particular cultivar (Yang et al., 1996; Yuan, 1997). Our earlier work (Yang et al., 2000a, 2001b,c) showed that water stress imposed during the grain®lling period of wheat and rice could enhance remobilization of pre-stored carbon reserves to the grains and accelerate grain ®lling. However, little is known whether and how the sink strength is involved in these processes. The objective of this study was to test the hypothesis that a controlled water de®cit during grain ®lling may enhance sink strength by regulating the key enzymes involved. The changes in activities of SuSase, AGPase, StSase and BE in rice grains and their relationship with grain ®lling were investigated. Another possible sucrose-cleaving enzyme in cereal grains, acid invertase (EC 3.2.1.26, AI), was also monitored for changes in its activity. 2. Materials and methods 2.1. Plant materials The experiment was conducted at a farm of Yangzhou University, Jiangsu Province, China (328300 N, 1198250 E) during rice growing season (May to October) of 1999, and repeated in 2000. Two lodgingresistant rice (O. sativa L.) cultivars currently used in local rice production, Wuyujing 3 (japonica) and Yangdao 4 (indica), were grown in the paddy ®eld. Seedlings were raised in the ®eld; sown during 10±11

May and transplanted during 10±11 June at a hill spacing of 0:20 m  0:16 m with two seedlings per hill. The soil was sandy loam (Typic ¯uvaquents, Etisols (US taxonomy)) with 24.5 g kg 1 organic matter and available N±phosphorus±potassium at 105, 33.5 and 66.0 mg kg 1, respectively. N (60 kg ha 1 as urea), phosphorus (30 kg ha 1 as single superphosphate) and potassium (40 kg ha 1 as KCl) were applied and incorporated before transplanting. N as urea was also applied at mid-tillering (40 kg ha 1) and at panicle initiation (25 kg ha 1). Both cultivars (50% of plants) headed on 20±22 August, and were harvested on 9±10 October. Except for drainage at the end of tillering (12±14 July), the ®eld was kept at 1±2 cm water level until 9 days after anthesis (DAA), when water-de®cit treatments were initiated. The temperatures, averaged per 10 days from anthesis (21±23 August) to harvest, were 26.9, 26.3, 25.2, 24.3, 23.2 and 22.6 8C. 2.2. Nitrogen and water stress treatments The experiment was a 2  2  2 (two cultivars, two levels of nitrogen, and two levels of soil moisture) factorial design with 8-treatment combinations. Each of the treatments had three plots as repetitions in a complete randomized block design. Plot dimension was 4:2 m  3:2 m and plots were separated by a ridge (40 cm in width) wrapped with plastic ®lm. Two levels of nitrogen treatments were applied at initial heading (10% of plants headed on 18±19 August). Half the plots were top-dressed with either 5 g N m 2 (normal nitrogen, NN) or 10 g N m 2 (high nitrogen, HN) as urea. From 9 DAA to maturity, two levels of soil water potential (csoil) were imposed on the plants of both NN and HN treatments. The well-watered (WW) treatment was kept at 1±2 cm water depth (csoil ˆ 0) in the ®eld by manually applying tap water every day, and the water-de®cit stressed (WS) was maintained csoil at 0.05 MPa. The csoil in the WS treatment was monitored with tension meters buried at 15±20 cm soil depth. Five tension meters were installed in each plot for monitoring purposes. Tension meter readings were recorded every 6 h from 06:00 to 18:00 h. When the reading dropped to the designated value, 100 l tap water per plot was added manually to the WS treatment. A rain shelter consisting of a steelframe covered with plastic sheet was used to protect the plots during rain.

J. Yang et al. / Field Crops Research 81 (2003) 69±81

2.3. Radioactive labeling At the boot stage (29 July for Wuyujing 3 and 30 July for Yangdao 6), 50 plants from each treatment were labeled with 14 CO2 . Flag leaves of main stems were used for labeling between 09:00 and 11:00 h on clear days with photosynthetically active radiation at the top of the canopy ranging between 1000 and 1100 mmol m 2 s 1. The whole ¯ag leaf was placed into a polyethylene chamber (25 cm length and 4 cm diameter) and sealed with tape. Six milliliters of air in the chamber was withdrawn and the same volume of mixed gas containing 14 CO2 was injected into the chamber (0.01 mol CO2 concentration at speci®c radioactivity of 14 C at 1.48 MBq l 1). The chamber was removed after 30 min. Labeled plants were harvested at 0 (50% anthesis), 9 (the initiation of water withholding), and from 12 to 42 DAA at 6-day intervals. Harvested plants were divided into leaf blades, stems (culms plus sheaths), and panicles. 14 C in the plants was assayed by the method described by Ge et al. (1994) with the following modi®cation: samples were dried in an oven at 80 8C to constant weight, ground into a powder, and then extracted by shaking in 80% (v/v) boiling ethanol. The residue was extracted in 2:1 of 60% (v/v) HClO4 to 30% (v/v) H2O2 for 4 h at 60 8C. The radioactivity of 14 C in both the extracted aliquots was counted using a liquid scintillation counter (Beckman Instruments Inc., Fullerton, CA). Radioactivity distribution to each part of the plant was expressed as a percentage of total radioactivity remaining in the aboveground portion of the plant. 2.4. Sampling The 180±200 panicles that headed on the same day were chosen and tagged for each plot. The ¯owering date and the position of each spikelet on the tagged panicles were recorded. About 10±12 tagged panicles from each plot were sampled at every 3-day interval from anthesis to 24 DAA and 6-day interval from 27 DAA to maturity. The sampled panicles were divided into two groups (®ve to six panicles each) as subsamples. Grains that developed from spikelets that ¯owered on the same day were removed. Half-sampled grains were frozen in liquid nitrogen for 1 min and then stored at 80 8C for enzymatic measurement.

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The other half of the grains was dried at 70 8C to constant weight for 72 h, and dehulled and weighed. The starch content in the grains was analyzed by the method of Yoshida et al. (1976). The processes of grain ®lling and starch accumulation in the grains were ®tted by Richards' (1959) growth equation as described by Zhu et al. (1988): Wˆ

A …1 ‡ B e

kt †1=N

(1)

Grain-®lling rate/starch accumulation rate (SAR) (G) was calculated using the derivative of Eq. (1): Gˆ

AKB e N…1 ‡ B e

kt

kt †…N‡1†=N

(2)

where W is the grain/starch weight (mg); A the ®nal grain/starch weight (mg); t the time after anthesis (days); and B, k, and N the coef®cients determined by regression. The active grain-®lling period was de®ned as that when W was from 5% (t1) to 95% (t2) of A. The average grain-®lling rate during this period was calculated from t1 to t2. The percentage of ripened grains and grain weight were determined from 50 plants (excluding the border ones) sampled randomly from each plot at maturity. The percentage of ripened grains was de®ned as the ripened grains (speci®c gravity 1.06) as a percentage of total spikelets. 2.5. Measurement of leaf water potential The measurement of leaf water potential was made at 2 h intervals at 19 and 20 DAA for both cultivars. Well-illuminated ¯ag leaves were chosen randomly for the measurement. A pressure chamber (Model 3000, Soil Moisture Equipment Corp., Santa Barbara, CA) was used for leaf water potential measurement with six leaves for each treatment. 2.6. Enzyme extraction and assays All chemicals and enzymes used for enzymatic measurement were from Sigma Chemical Company (St. Louis, MO). All enzyme assays were optimized for substrate concentration and pH and were within the linear phase with respect to incubation time and protein concentration. Protein content was determined

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J. Yang et al. / Field Crops Research 81 (2003) 69±81

according to Bradford (1976), using bovine serum albumin as a standard. Enzyme activities were expressed on a per grain basis. The method for preparation of enzyme extracts was modi®ed from Nakamura et al. (1989). Brie¯y, 45±50 dehulled and frozen grains were homogenized with a pestle in a pre-cooled mortar that contained 8 ml frozen extraction medium: 100 mM HEPES±NaOH (pH 7.6), 8 mM MgCl2, 5 mM dithiothreital, 2 mM EDTA, 12.5% (v/v) glycerol, and 5% (w/v) insoluble polyvinylpyrrolidone 40. After being ®ltered through four layers of cheesecloth, the homogenate was centrifuged at 12,000  g for 10 min, and the supernatant was used for the enzyme assay. SuSase was assayed in the cleavage direction and analyzed as described by Ranwala and Miller (1998). Soluble and insoluble AI, and soluble and insoluble StSase activities were determined according to the methods of Zinselmeier et al. (1995) and Schaffer and Petreikov (1997), respectively. AGPase and BE were assayed by the method of Nakamura et al. (1989). 2.7. Statistical analysis The results were analyzed for variance using SAS statistical analysis package (version 6.12, SAS Institute, Cary, NC). Data from each sampling date were analyzed separately. Means were tested by least signi®cant difference at P0.05 level (LSD0.05). Linear regression was used to evaluate the relationship between enzymatic activities and SAR in the grains. 3. Results 3.1. Leaf water potential Fig. 1 illustrates the diurnal changes in ¯ag leaf water potential at 10±11 days after withholding water. Both cultivars exhibited a similar pattern of leaf water potential changes. At NN level, the leaf water potential ranged from 0.05 MPa at pre-dawn (06:00 h) to 0.72 MPa at midday (12:00 h) for the plants under WW treatments. It was greatly reduced for the plants under WS treatments during the day, and reached 1.25 to 1.30 MPa at midday. Plants under HN

had lower midday leaf water potential than those under NN, even though csoil was kept at the same level, suggesting that leaves with better N nutrition lost more water, probably because of high leaf conductance. However, the differences in leaf water potential in the early morning between WS and WW treatments were very small, either at NN or HN, indicating that plants subjected to WS could rehydrate overnight. 3.2. Fixed carbon partitioning and grain-®lling rate Water stress facilitated the reallocation of preanthesis assimilates from the stems to grains. Fig. 2 shows the disappearance of pre-anthesis assimilated 14 C in the stems and its appearance in the grains during grain ®lling. At the start of water withholding (9 DAA), about 75% of 14 C fed to the ¯ag leaves at the booting stage was partitioned in the stems, and about 10% in the grains. After 12 days (21 DAA), 14 C in the stem was reduced to 35±37% under WS and 46± 48% under WW treatments at NN level. In contrast to that observed in the stem, the 14 C in the grains increased by 44±46% under WS and only 25±27% under WW treatments at 21 DAA. At maturity, 65± 70% of 14 C pre-stored in the stem was reallocated to grains under WS, 35±40% higher than the amount under WW treatments at NN level. Compared with NN, HN reduced reallocation of 14 C from the stem to grains when csoil was the same. Water stress greatly accelerated starch accumulation in grains from 9 to 27 DAA (Fig. 3). Eighty-nine to 96% of ®nal starch weight in the grains was accumulated under WS treatments during this period, and only 64±78% under WW treatments in the same period. HN slowed the starch accumulation either under WW or WS treatments. Water stress increased grain-®lling rate and shortened grain-®lling period (Table 1). The active grain-®lling period was shortened by 2.9±5.5 days at NN and 5.7±7.4 days at HN, and grain-®lling rate increased by 0.18±0.29 mg per grain per day at NN and 0.31±0.37 mg per grain per day at HN, respectively, compared with their respective WW treatments. The percentage of ripened grains and grain weight were not signi®cantly different between WS and WW treatments at NN. However, they were signi®cantly greater under WS than under WW treatments at HN (Table 1), implying that the

J. Yang et al. / Field Crops Research 81 (2003) 69±81

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Fig. 1. Diurnal changes of leaf water potentials of the japonica cultivar Wuyujing 3 (A) and indica cultivar Yangdao 4 (B). Symbols WW and WS are well-watered and water-de®cit stressed during grain ®lling, and NN and HN indicate normal and high levels of N application at heading time, respectively. Measurements were made on the ¯ag leaves 19 DAA for Wuyujing 3 and 20 DAA for Yangdao 4. Vertical bars represent S.E. of the mean (n ˆ 6) where these exceed the size of the symbol.

gain from accelerated grain-®lling rate outweighed the possible loss of photosynthesis as a result of a shortened grain-®lling period when subjected to water stress during grain ®lling. 3.3. Changes in SuSase and AI activities The activity of SuSase, on a per grain basis, is much higher than that of AI (Fig. 4A±D). The average SuSase activity was 78.5 nmol per grain min 1 during rapid starch accumulation period (12±27 DDA, refer to Fig. 3), whereas soluble and insoluble AIs in this

period were 15.4 and 4.3 nmol per grain min 1, respectively, indicating that SuSase is a predominant enzyme responsible for sucrose cleavage in rice grains. SuSase activity in the grains was substantially enhanced by water stress during the ®rst 9 days after water withholding (9±18 DAA), reached its peak at 15±18 DAA, and sharply decreased thereafter (Fig. 4A and B). Its activity was lower under WS than under WW treatments at mid and later grain®lling stages (21 DAA afterward). SuSase activity was higher in NN grains than in HN grains at early

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Fig. 2. Changes of 14 C partitioning in the stems (A and B) and grains (C and D) of the japonica cultivar Wuyujing 3 (A and C) and indica cultivar Yangdao 4 (B and D). Treatment details are the same as in Fig. 1. The 14 C was fed to the ¯ag leaves at booting stage. Arrows ®gure indicate the start of withholding water. Vertical bars represent S.E. of the mean (n ˆ 6) where these exceed the size of the symbol.

grain-®lling stages when csoil was the same. During mid- and late grain-®lling periods, the differences were reversed and HN grains had higher SuSase activity than NN grains.

The changing pattern of soluble AI activity was somewhat similar to that of SuSase (Fig. 4C and D). However, soluble AI activity was less enhanced by water stress. Insoluble AI activity in the grains

Table 1 Grain-®lling rate and grain yield of rice subjected to various N and soil moisture treatmentsa Cultivars

Nitrogen applied

Water-deficit treatment

Active grain-filling period (days)

Grain-filling rate (mg per grain per day)

Ripened grains (%)

Grain weight (mg grain 1)

Wuyujing 3

NN NN HN HN

WW WS WS WS

19.7 17.0 24.8 19.1

b c a b

1.21 1.39 0.91 1.28

c a d b

90.8 90.2 84.2 94.2

b b c a

26.2 26.3 25.1 27.1

b b c a

Yangdao 4

NN NN HN HN

WW WS WW WS

23.9 18.4 28.6 21.2

b d a c

1.02 1.31 0.82 1.14

c a d b

80.5 78.9 74.6 82.5

ab b b a

27.1 26.8 26.1 26.9

a a b a

a NN and HN indicate normal and high levels of nitrogen application at heading time. WW and WS are well-watered and water-de®cit stress treatments during grain ®lling, respectively. Active grain-®lling period and grain-®lling rate were calculated according to Richards (1959) equation. Values of grain weight and percentage of ripened grains were means of 148±156 plants. Letters indicate statistical signi®cance at P0.05 within the same cultivar.

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Fig. 3. Starch accumulation process (A and B) and SAR (C and D) in the grains of the japonica cultivar Wuyujing 3 (A and C) and indica cultivar Yangdao 4 (B and D). Treatment details are the same as in Fig. 1. SAR was calculated according to Richards (1959) equation. Arrows indicate the start of withholding water. Vertical bars in A and B represent S.E. of the mean (n ˆ 3) where these exceed the size of the symbol.

changed little during the grain-®lling period and differences were not signi®cant between WW and WS or between NN and HN treatments (Fig. 4E and F). The activity of SuSase, but not that of AI, paralleled starch increases in the grains during the rapid starch accumulation period (refer to Fig. 3). Regression analysis demonstrated that SuSase activity was positively correlated with SAR with r ˆ 0:81 ( P < 0:01, Fig. 5A), whereas neither of the soluble and insoluble AIs was positively correlated (r ˆ 0:18 and 0.086, P > 0:05, Fig. 5B and C). 3.4. Changes in AGPase, StSase and BE activities For all the treatments, AGPase, soluble StSase and BE activities increased initially and declined after reaching a maximum. The peak activity values of

the three enzymes were greater and declined faster under WS than under WW treatments and less at HN than at NN when csoil was the same (Fig. 6A±F). However, the three enzymes exerted somewhat different activity pro®les with respect to the time taken to reach the peak and the effect of water stress. AGPase activity showed a peak at 12±15 DAA, which corresponded to the initial stage of rapid starch accumulation in the grain (refer to Fig. 3). Soluble StSase and BE activities appeared with their peaks at 15±18 and 18±21 DAA, respectively, when starch was being rapidly accumulated in grains. Water stress enhanced to a smaller level soluble StSase activities, but greatly enhanced BE activity during the ®rst nine days after water withholding. AGPase activity was little affected by the WS (Fig. 6). Insoluble StSase activity in the grains was very low, and ranged from 0.3 to 0.8 nmol per grain min 1. There were no signi®cant

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Fig. 4. Changes in activities of sucrose synthase (A and B), soluble AI (C and D) and insoluble invertase (E and F) in the grains of the japonica cultivar Wuyujing 3 (A, C and E)) and indica cultivar Yangdao 4 (B, D and F). Treatment details are the same as in Fig. 1. Arrows indicate the start of withholding water. Vertical bars represent S.E. of the mean (n ˆ 3) where these exceed the size of the symbol.

differences between WW and WS treatments (data not shown). Changes in BE activity during the rapid starch accumulation period corresponded well with starch levels in the grain (refer to Fig. 3). The correlation between BE activity and SAR was positive and statistically signi®cant (r ˆ 0:84,  P < 0:01, Fig. 7C). Soluble StSase activity was also correlated with SAR (r ˆ 0:69,  P < 0:01, Fig. 7B). No signi®cant correlation was observed between AGPase activity and SAR (r ˆ 0:084, P > 0:05, Fig. 7A).

4. Discussion Our results demonstrated that if water de®cit during grain ®lling is properly controlled, so that plants can rehydrate overnight (Fig. 1), remobilization of prestored carbon in the stems can be remarkably enhanced (Fig. 2) and grain-®lling rate greatly increased (Fig. 3, Table 1). The gain from the enhanced remobilization and accelerated grain-®lling rate may outweigh the possible loss of photosynthesis due to shortened grain-®lling period and increased

J. Yang et al. / Field Crops Research 81 (2003) 69±81

Fig. 5. Relationship of SAR in grains with the activities of sucrose synthase (A), soluble AI (B) and insoluble AI (C) during rapid starch accumulation period (12±27 DAA). Data are from Figs. 3 and 4. Correlation coef®cients (r) are calculated and ** represent statistical signi®cance at the P0.01 level.

grain yield in the case where grain ®lling is slowed by the heavy use of N-fertilizers or the adoption of high lodging-resistant cultivars that stay ``green'' for a longer period. It is generally accepted that grain-®lling rate is closely associated with sink strength (Venkateswarlu

77

and Visperas, 1987; Cao et al., 1992; Liang et al., 2001). During grain-®lling period, rice grains are strong carbohydrate sinks (Cao et al., 1992). The sink strength can be described as the product of sink size and sink activity (Venkateswarlu and Visperas, 1987). Sink size is a physical restraint that includes cell number and cell size. Sink activity is a physiological restraint that includes multiple factors and key enzymes involved in carbohydrate utilization and storage (Wang et al., 1993). Our early work (Yang et al., 2000b) showed that a mild water stress imposed at 9 DAA till maturity had no serious effect on cell number and cell size of rice endosperm. In this study, differences in ripened grain percentage and grain weight were not signi®cantly between WS and WW treatments even at a NN level (Table 1). We speculate that the grain sink size should not be affected by the water stress in the present experiment, but the sink activity should be substantially enhanced. There are reports that both SuSase and AI are involved in sucrose cleavage in sink tissue and their activities are regarded as biochemical markers of sink strength (Wang et al., 1993; Ranwala and Miller, 1998). The results showed that the activity of SuSase in rice grains was much higher than those of both soluble and insoluble AIs, and water stress enhanced the former more than it did the latter at the early water withholding stage (Fig. 4). SuSase activity was positively correlated with SAR in grains during the rapid starch accumulation period, whereas neither the soluble nor insoluble AIs was positively correlated (Fig. 5). A probable explanation is that SuSase is predominant in sinks accumulating reserve carbohydrates, whereas invertase is mainly present in tissues in which active cell elongation is occurring (Sung et al., 1988; Ranwala and Miller, 1998). The results suggest that the enhanced carbon remobilization and grain-®lling rate by a water de®cit imposed during grain-®lling period is attributed to, or links to an increase in sink strength by regulating SuSase activity in rice grains. Water stress greatly enhanced BE activity during the ®rst nine days after water withholding (Fig. 6C). BE activity paralleled starch increases in the grains and positively correlated with SAR in grains during rapid starch accumulation period (Fig. 7C). Though soluble StSase activity was also associated with starch levels, it was less enhanced by WS treatments (Fig. 6B) and less closely correlated with SAR, when compared

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Fig. 6. Changes in activities of adenine diphosphoglucose pyrophosphorylase (AGPase, A and B), soluble starch synthase (C and D) and starch BE (E and F) in the grains of the japonica cultivar Wuyujing 3 (A, C and E) and indica cultivar Yangdao 4 (B, D and F). Treatment details are the same as in Fig. 1. Arrows indicate the start of withholding water. Vertical bars represent S.E. of the mean (n ˆ 3) where these exceed the size of the symbol.

to BE (Fig. 7B). It is proposed that BE is the sole enzyme capable of forming a-1,6-linked branches on already synthesized and/or elongating amylose molecules (Preiss et al., 1991). BE is also considered to activate StSase by providing it with the non-reducing end needed for acceptance of a newly synthesized glucan unit (Preiss, 1988) thereby plays a key role in starch production in rice endosperm (Nakamura et al., 1989; Nakamura and Yuki, 1992). In this study, close correlation of BE activity with SAR suggests that accelerated grain-®lling rate under water stress is mainly associated with the enhanced BE activity.

Water stress appeared to have little effect on AGPase activity (Fig. 6A). No signi®cant correlation was observed between its activity and SAR (Fig. 7A). A similar result on wheat anthers was also observed by Dorion et al. (1996). AGPase is commonly regarded as the rate-limiting enzyme in starch biosynthesis (Preiss, 1988). However, the evidence provided by Stitt (1994) and Smith et al. (1995) suggests that AGPase may not be the only step at which biosynthesis is regulated in non-photosynthetic tissue. It is notable that the peak of AGPase activity corresponds with the initial of rapid starch accumulation (Figs. 3 and 6A).

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Fig. 7. Relationship of SAR in grains with the activities of adenine diphosphoglucose pyrophosphorylase (AGPase) (A), starch synthase (StSase) (B) and starch BE (C) during rapid starch accumulation period (12±27 DAA). Data are from Figs. 3 and 6. Correlation coef®cients (r) are calculated and ** represent statistical signi®cance at the P0.01 level.

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It is suggested that AGPase may play an important role in initiating starch accumulation in rice grains. Obviously, the role of AGPase in starch synthesis in rice grains still needs to be elucidated. 5. Conclusion If water de®cit during grain ®lling of rice is properly controlled, so that plants can rehydrate overnight, remobilization of pre-stored carbon in the stems can be remarkably enhanced and grain-®lling rate greatly increased. WS-increased remobilization and grain®lling rate are mainly attributed to, or associated with enhanced sink strength by regulating sucrose synthase and starch BE activities in the grains. Acknowledgements This research was funded by the FRG of the Hong Kong Baptist University, RGC of the Hong Kong University Grants Council, AOE Research Foundation of the Chinese University of Hong Kong, the National Natural Science Foundation of China (Project No. 39970424) and the State Key Basic Research and Development Plan (G 1999011704). References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248±254. Cao, X., Zhu, Q., Yang, J., 1992. Classi®cation of source±sink types in rice varieties with corresponding cultivated ways. In: Min, S. (Ed.), Prospects of Rice Farming for 2000. Zhejiang Publishing House of Science and Technology, Hangzhou, pp. 360±365. Dorion, S., Lalonde, S., Saini, H.S., 1996. Induction of male sterility in wheat by meiotic-state water de®cit is preceded by a decline in invertase activity and changes in carbohydrate metabolism in anthers. Plant Physiol. 111, 137±145. Ge, C., Gong, J., Lou, S., Zhang, H., 1994. Tracer kinetics research on the variation of transporting velocity of photosynthate in sheath and stem in rice. Acta Agric. Nuc. Sin. 8, 33±40. Kato, T., 1995. Change of sucrose synthase activity in developing endosperm of rice cultivars. Crop Sci. 35, 827±831. Liang, J., Zhang, J., Cao, X., 2001. Grain sink strength may be related to the poor grain ®lling of indica±japonica rice (Oryza sativa) hybrids. Physiol. Plant. 112, 470±477.

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