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First published online July 2, 2004; 10.1104/pp.104.041038 Plant Physiology 135:1621-1629 (2004) © 2004 American Society of Plant Biologists Activities of Key Enzymes in Sucrose-to-Starch Conversion in Wheat Grains Subjected to Water Deficit during Grain Filling1College of Agriculture, Yangzhou University, Yangzhou, Jiangsu, China 225009 (J.Y., Z.W., G.X., Q.Z.); and Department of Biology, Hong Kong Baptist University, Hong Kong, China (J.Z.)
This study tested the hypothesis that a controlled water deficit during grain filling of wheat (Triticum aestivum) could accelerate grain-filling rate through regulating the key enzymes involved in Suc-to-starch pathway in the grains. Two high lodging-resistant wheat cultivars were field grown. Well-watered and water-deficit (WD) treatments were imposed from 9 DPA until maturity. The WD promoted the reallocation of prefixed 14C from the stems to grains, shortened the grain-filling period, and increased grain-filling rate or starch accumulation rate (SAR) in the grains. Activities of Suc synthase (SuSase), soluble starch synthase (SSS), and starch branching enzyme (SBE) in the grains were substantially enhanced by WD and positively correlated with the SAR. ADP Glc pyrophosphorylase activity was also enhanced in WD grains initially and correlated with SAR with a smaller coefficient. Activities of granule-bound starch synthase and soluble and insoluble acid invertase in the grains were less affected by WD. Abscisic acid (ABA) content in the grains was remarkably enhanced by WD and very significantly correlated with activities of SuSase, SSS, and SBE. Application of ABA on well-watered plants showed similar results as those by WD. Spraying with fluridone, an ABA synthesis inhibitor, had the opposite effect. The results suggest that increased grain-filling rate is mainly attributed to the enhanced sink activity by regulating key enzymes involved in Suc-to-starch conversion, especially SuSase, SSS, and SBE, in wheat grains when subjected to a mild water deficit during grain filling, and ABA plays a vital role in the regulation of this process.
Starch in the endosperm of wheat (Triticum aestivum) is the major form of carbon reserves and comprises 65% to 75% of the final dry weight of the grain (Housley et al., 1981  ; Dale and Housley, 1986; Hurkman et al., 2003). Grain filling is therefore mainly a process of starch biosynthesis and accumulation. It is generally accepted that four enzymes may play a key role in this process: Suc synthase (SuSase; EC 2.4.1.13), ADP Glc pyrophosphorylase (AGPase; EC 2.7.7.27), starch synthase (StSase; EC 2.4.1.21), and starch branching enzyme (SBE; EC 2.4.1.18; Hawker and Jenner, 1993; Ahmadi and Baker, 2001; Hurkman et al., 2003). SuSase catalyses the cleavage of Suc, the main transported form of assimilates in wheat plants (Fisher and Gifford, 1986), to form UDP-Glc and Fru, which is thought to be the first step in the Suc-to-starch conversion. Its activity is considered to be linked to sink strength in the developing rice (Oryza sativa) grain and tomato (Lycopersicon esculentum) fruit (Sun et al., 1992; Wang et al., 1993; Kato, 1995). AGPase produces ADP-Glc, the primer of the starch chain (Smith and Denyer, 1992), and is regarded as the rate-limiting enzyme in starch biosynthesis (Preiss, 1988). StSase, composed of both soluble and granule-bound isoforms, elongates the amylose and amylopectin chains (Déjardin et al., 1997). Soluble StSase (SSS) activity is reported to be positively correlated with the rate of starch synthesis in wheat grains (Keeling et al., 1993). SBE forms branches on the polymers. It cleaves  -1,4 bonds on both amylose and amylopectin molecules and reattaches the released glucan segments to the same or another glucan chain through the formation of -1,6 linkages (Hurkman et al., 2003). Its activity is closely associated with the increase in starch content during the development of rice endosperm (Nakamura et al., 1989; Nakamura and Yuki, 1992).
Extensive studies have been done on the effects of heat stress on the activities of enzymes involved in Suc-to-starch metabolism in cereals (Caley et al., 1990
Abscisic acid (ABA) is generally regarded as a very sensitive signal produced during water stress (Zhang and Davies, 1990a
Leaf Water Potential Figure 1 illustrates the progression of leaf water potentials during the first 22 d after withholding water. When plants were well-watered (WW), mid-day (11:30 AM) leaf water potentials decreased gradually during grain filling, from –0.96 million Pa (MPa) at the beginning (9 DPA) to –1.43 to –1.55 MPa on 22 d after withholding water (31 DPA). The water-deficit (WD) treatment substantially reduced mid-day leaf water potentials from –0.98 MPa at 9 DPA to –2.31 to –2.50 MPa at 31 DPA. However, the differences in predawn (6 AM) leaf water potentials between WW and WD plants were insignificant (Fig. 1), indicating that plants subjected to water deficit could rehydrate overnight.
Carbon Remobilization and Grain-Filling Rate At early grain-filling stage (0–12 DPA), the main form of nonstructural carbohydrate in the stem of wheat was fructan, Suc, Glc, and Fru (56%, 25%, 10%, and 9%, respectively), and the activities of fructan exohydrolase and Suc phosphate synthase in the stem were substantially enhanced by WD (data not shown). The WD facilitated the reallocation of preanthesis assimilates from the stems to grains. Figure 2 shows the disappearance of preanthesis-assimilated 14C in the stems and its appearance in the grains during grain filling. At the start of water withholding (9 DPA), about 70% of 14C fed to the flag leaves at the booting stage was partitioned in the stems and about 5% in the grains. After 24 d (33 DPA), 14C in the stem was reduced by 23% to 34% under WD and 50% to 61% under WW treatments. Opposite to that observed in the stem, the 14C in the grains increased by 39% to 50% for WD plants and only 9% to 18% for WW ones at 33 DPA. In comparison, application of a high amount of N (HN) reduced the 14C reallocation into the grains (Fig. 2).
The WD greatly accelerated starch accumulation in grains from 9 to 27 DPA at normal amount of N (NN) and from 12 to 33 DPA at HN (Fig. 3). Eighty-one percent to 92% of the final starch weight in WD grains was accumulated during this period, and only 44% to 52% for WW grains in the same period. HN slowed the starch accumulation either for WW or WD plants. A similar observation was made on grain-filling rate (Table I). The WD substantially increased the grain-filling rate and shortened the grain-filling period at both NN and NH. The active grain-filling period was shortened by 8 d at NN and 10 d at HN, and grain-filling rate increased by 0.54 mg per day per kernel at NN and 0.74 mg per day per kernel at HN, respectively, compared with their respective WW treatments.
The final grain weight was not significantly different between the WW and WD treatments when NN was applied (Table I). However, it was significantly increased under HN plus WD treatments. A similar result was obtained with grain yield because only the kernel weight, rather than the spike number per m2 or grain number per spike, was influenced by the WD during grain filling in this experiment (Table I).
The activities of the five enzymes examined in relation to Suc-to-starch conversion in wheat grains exhibited variable responses with time, soil moisture, and N levels. SuSase activity (assayed in cleavage direction) in WW grains was increased from 9 to 27 DPA and then decreased with grain development (Fig. 4A). It was substantially enhanced by WD during the first 9 to12 d after withholding water, reached its peak 18 and 21 DPA at NN and HN, respectively, and sharply declined thereafter, in good agreement with the starch accumulation rate (SAR; refer to Fig. 3B).
Irrespective of NN and HN treatments, activities of both soluble and insoluble AI in WW grains were little changed from 9 to 18 DPA and decreased afterward (Fig. 4, B and C). They were enhanced by WD, with soluble AI enhanced more than insoluble AI. SuSase activity, on a per grain basis, was much higher than those of AI during the rapid accumulation period of starch in the grain (9–27 DPA at NN and 12–33 DPA at HN, respectively, for WD plants and 12–39 DPA for WW plants; refer to Fig. 3), indicating that SuSase is a predominant enzyme responsible for Suc cleavage in wheat grains. Very similar to SuSase activities, SSS and SBE activities were markedly increased in WD grains during the first 9 to 12 d after withholding water (Fig. 5, B and D). The changes in activities of both SSS and SBE were consistent with SAR (refer to Fig. 3). By contrast, granule-bound starch synthase (GBSS) activity was little affected by either the WD or N treatments (Fig. 5C).
AGPase activity exhibited a peak at 15 to18 DPA in WD grains and 21 to 27 DPA in WW grains (Fig. 5A). The peak appeared just before or corresponded to the maximum SAR. The activity in WD grains was greater initially and declined faster after reaching a maximum when compared with that in WW grains. With the exception of GBSS activity, HN reduced activities of all the enzymes at early and mid-grain-filling stages but slowed down their declines at the late filling stage when  soil was the same.
As shown in Figure 6, ABA content in the grains was very low at early grain-filling stage and remarkably enhanced by WD at both NN and HN. At early and mid-grain-filling stages, the grain with NN contained more ABA than those with HN treatments when soil water potential was the same. The change in ABA content in the grains was paralleled with SAR (refer to Fig. 3), and they were very significantly correlated (r = 0.99**, P = 0.01).
Correlations of Enzyme Activities with ABA Content and Starch Accumulation Rate During the rapid accumulation period of starch in the grain, the correlations of activities of the five enzymes examined with ABA content and SAR were analyzed (Table II). Activities of SuSase, SSS, and SBE were positively and very significantly correlated with SAR (r = 0.92** to 0.97 **, P = 0.01). AGPase activity was also correlated with SAR with r = 0.69* (P = 0.05), whereas neither of the soluble or insoluble AI nor GBSS was significantly correlated (r = 0.35–0.48, P > 0.05). Very similar correlations were observed between the activities of enzymes and ABA content in the grains (Table II), suggesting that ABA may play an important regulative role in enzyme activities.
Effects of Exogenous ABA and Fluridone When ABA was applied to HN plus WW plants at early grain-filling stage (9–13 DPA), the activities of SuSase, SSS, and SBE and starch accumulation in the grains were very significantly increased (Table III). Effects of ABA application on AGPase and soluble AI activities varied with the determination date. Supplemental ABA had no significant effects on the activities of insoluble AI and GBSS. Opposite to ABA, spraying with fluridone, an inhibitor of ABA synthesis, significantly reduced activities of SuSase, SSS, SBE, and AGPase and starch content in the grains (Table III). The final grain weight was 46.1 g and 26.6 g, respectively, for ABA- and fluridone-treated plants, which was 129% and 75% of that (35.6 g) for the control, respectively.
Water stress imposed during the grain-filling period of wheat, especially at the early filling stage, usually results in a reduction in grain weight and leads to reduced grain yield (Aggarwal and Sinha, 1984 ; Nicolas et al., 1985b; Kobata et al., 1992; Zhang et al., 1998). We found, however, that if WD during grain filling was controlled properly, plants were able to rehydrate overnight (Fig. 1). A benefit from such a WD was that it could enhance remobilization of carbon reserves from vegetative tissues to the grains (Fig. 2) and accelerate SAR and the grain-filling rate (Fig. 3; Table I). The grain weight and grain yield would not necessarily be reduced when NN was applied (Table I). Moreover, the grain weight and grain yield could be increased when excessive N fertilizer was applied (Table I).
The discrepancies between our results and previous reports (Aggarwal and Sinha, 1984
It is generally accepted that grain-filling rate in cereals is mainly determined by sink strength (Venkateswarlu and Visperas, 1987
It was hypothesized that high levels of enzymes involved in the breakdown of Suc in the sink would increase sink capacity by lowering the local concentration of Suc, thereby generating a gradient that allows further unloading of Suc from phloem (Wardlaw, 1968
WD enhanced activities of AGPase, SSS, and SBE at initial or at early and mid-grain-filling stages (Fig. 5). The activities of the three enzymes were positively correlated with SAR (Table II), suggesting that all of the three enzymes in wheat grains play an important role in starch synthesis. We observed that GBSS activity was neither enhanced by the WD nor significantly correlated with SAR (Table II). A probable explanation is that SSS is thought to be responsible for generating polymers that are the substrates for amylopectin synthesis via SBE (Smith and Denyer, 1992
It is worth noting that SBE was highly correlated with SAR with the greatest coefficient (r = 0.97) among all the enzymes examined in this experiment (Table II). The result may probably be explained by the fact that SBE is the sole enzyme capable of forming
The results that a mild WD imposed during grain-filling period could enhance activities of key enzymes in Suc-to-starch pathway were also observed on other wheat cultivars and rice (Yang et al., 2001a
Our results demonstrate that if a WD stress during the grain filling of wheat is controlled properly so that plants can rehydrate overnight, remobilization of prestored carbon from vegetative tissues to the grains can be substantially enhanced and SAR or grain-filling rate be accelerated. The increased remobilization and grain-filling rate are mainly attributed to, or at least linked to, the enhanced sink activity by regulating key enzymes involved in Suc-to-starch pathway, especially SuSase, SSS, and SBE, in the grains when subjected to the WD. ABA plays a vital role in the regulation of this process.
Plant Materials and Cultivation
The experiment was conducted at Yangzhou University farm, Yangzhou, China (32°30'N, 119°25'E) from November 2002 to June 2003. Two highly lodging-resistant cultivars of semi-winter wheat (Triticum asetivum), cv Yangmai 158 and cv Yangmai 11, currently used in local production, were grown in the field. The sowing date was November 2, and the plant density was adjusted to 150 plants m–2 at three-leaf age. The soil of the field was sandy loam soil (Typic fluvaquents, Entisols; U.S. taxonomy) that contained organic matter at 2.42% and available N-phosphorus-potassium at 110, 36.5, and 70.4 mg kg–1, respectively. On the day of sowing, 14 g N m–2 as urea and 4 g phosphorus m–2 as single superphosphate were applied to the soil. On 30 d after sowing (DAS) and 112 DAS, 6 g and 5 g N m–2 as urea were top dressed, respectively. The soil water content was maintained close to field capacity (soil water potential,
The experiment was a 2 x 2 x 2 (two cultivars, two levels of N, and two levels of soil moisture) factorial design with eight treatments. Each of the treatments had three plots as repetitions in a complete randomized block design. Plot dimension was 3 m x 4 m, and plots were separated by a ridge (20 cm in width) wrapped with plastic film. Two levels of N treatments were applied at heading (50% of plants headed). One-half of plots were top dresses with either 3 g N m–2 (NN) or 8 g N m–2 (HN) as urea. From 9 DPA (173 DAS for both cultivars) until maturity, two levels of
At the boot stage (144 DAS for Yangmai 158 and 146 DAS for Yangmai 11), 60 plants from each treatment were labeled with 14CO2. Flag leaves of main stems were used for the labeling between 9 AM to 11 AM on a clear day with photosynthetically active radiation at the top of the canopy ranging between 1,000 and 1,100 µmol m–2 s–1. The whole flag leaf was placed into a polyethylene chamber (25-cm length and 4-cm diameter) and sealed with tape and plasticine to maintain a gas-tight seal. Ten milliliters of air in the chamber was drawn out, and the same volume of gas was injected into the chamber, which contained 0.015 mol L–1 CO2 at a specific radioactivity of 14C of 2.21 MBq L–1. The chamber was removed after 1 h. Labeled plants were harvested at 0 DPA (50% anthesis) and from 9 (the initiation of water withholding) to 54 DPA at 6-d intervals, respectively. Each plant was divided into leaf blades, culms plus sheaths, kernels, and other parts on a spike (rachis + palea + lemma + glume). Samples were dried at 80°C to constant weight, ground into powder, and then extracted by shaking for 30 min 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°C. The radioactivity of 14C in both the extracted aliquots was counted using a liquid scintillation counter (Beckman Instruments, Fullerton, CA), and the data were presented in composite averages. Radioactivity distribution in each part of the plant was expressed as a percentage of total radioactivity remaining in the aboveground portion of the plant.
A total of 150 spikes that headed on the same day were chosen and tagged for each plot. Ten tagged spikes from each plot were sampled at 3-d intervals from anthesis to 21 DPA and 6-d intervals from 27 DPA to maturity. All grains from each spikelet were removed. Half of the sampled grains was frozen in liquid nitrogen for 2 min and then stored at –80°C for enzymatic and ABA measurements. The other half of the grains was dried at 70°C to constant weight and weighed. The starch content in the grains was analyzed by the method of Yoshida et al. (1976)
Both grain-filling rate and SAR (G) were calculated as the derivative of Equation 1:
Plants (except border) from a 4-m2 site from each plot were harvested at maturity for the determination of grain yield. Yield components, i.e. the spikes per square meter, grain number per spike, and grain weight, were determined from plants harvested from a 1-m2 site (excluding the border plants) randomly sampled from each plot.
Leaf water potentials of flag leaves were measured on clear days at predawn (6 AM) and midday (11:30 AM) on 0, 3, 6, 10, 14, 18, and 22 d after withholding water. Well-illuminated flag leaves were chosen randomly for such measurements. A pressure chamber (model 3000; Soil Moisture Equipment, Santa Barbara, CA) was used for leaf water potential measurement with six leaves for each treatment.
All chemicals and enzymes used for enzymatic measurement were from Sigma (St. Louis). All enzyme assays were optimized for pH and substrate concentration and were within the linear phase with respect to incubation time and protein concentration. Protein content was determined according to Bradford (1976)
The method for SuSase and AI extraction was modified from Ranwala and Miller (1998) For SuSase extraction, grains were homogenized with a mortar and pestle in 100 mM HEPES (pH 7.5) containing 10 mM isoascorbate, 3 mM MgCl2, 5 mL dithiothreitol (DTT), 2 mL EDTA, 5% (v/v) glycerol, 3%(w/v) polyvinylpyrrolidone, and 0.01% Triton X-100. After centrifugation at 15,000g for 30 min, the supernatant was desalted on a Sephadex G-25 column, and the proteins were eluted by the reaction buffer that contained 50 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM EDTA, and 3 mM DTT.
The extraction procedure for AGPase, StSase, and SBE was according to Nakamura et al. (1989)
The enzyme activities were determined as described previously: SuSase (in cleavage direction; Ranwala and Miller, 1998
The methods for extraction and purification of ABA [(±)-ABA] were modified from those described by Bollmark et al. (1988)
The mouse monoclonal antigen and antibody against ABA, and IgG-horse radish peroxidase (IgG-HRP) used in ELISA were produced at the Phytohormones Research Institute, China Agricultural University, China (see He, 1993
Plants were grown in eight cement tanks in open field conditions. Each tank (0.3-cm height, 1.6-m width, and 8.8-m length) was filled with sandy loam soil with the same nutrient contents as the field soil. The sowing date and cultivation were the same as the field experiment. A HN plus WW treatment was conducted as described above. Starting 9 DPA, either 20 x 10–6 M (±)-ABA (Sigma) or 20 x 10–6 M fluridone (Fluka, NJ; Riedel-de Haën, Seelze, Germany), an inhibitor of ABA synthesis, were sprayed at the rate of 800 mL m–2 on the top of plants (spikes) for 5 d. Both ABA and fluridone were applied between 4 PM and 5 PM on each day. All the solutions contained ethanol and Tween 20 at final concentrations of 0.1% (v/v) and 0.01 (v/v), respectively. Control plants were sprayed with the same volume of deionized water containing same concentrations of ethanol and Tween 20. Each treatment was an area of 2.4 m2 with four replications. ABA content, enzymatic activities, and starch content in the grains were determined 3, 6, and 9 d after the chemical treatments (15, 18, and 27 DPA). Measurement methods were the same as described above. Fifty plants from each treatment were harvested at maturity for the determination of final grain weight.
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 LSD at P0.05 level (LSD0.05). Linear regression was used to evaluate the relationships of enzymatic activities with ABA content and SAR in the grains. Since the two cultivars behaved the same, the data are presented as an average between the two cultivars. This experiment was also conducted under pot-grown conditions at the same time as the field-grown, and results from the pot experiment were very similar. Only the field experiment was reported in this paper because of limited space. Received February 14, 2004; returned for revision April 12, 2004; accepted April 12, 2004.
1 This work was supported by the Research Grant Council of Hong Kong (RGC 2052/00M), by the Area of Excellence for Plant and Fungal Biotechnology in the Chinese University of Hong Kong, and by the State Key Basic Research and Development Plan (grant no. G1999011704).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041038. * Corresponding author; e-mail jzhang{at}hkbu.edu.hk; fax 852–3411–5995.
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), NN + WD (
), HN + WW (
), and HN + WD (
). Measurements were made on the flag leaves at predawn (6 AM, dashed lines) and at midday (11:30 AM, solid lines). Vertical bars represent ±SE of the mean (n = 12) where these exceed the size of the symbol.
-mercaptoethanaol, 10 mM isoascorbate, and 1 mM phenylmethyl-sulfonyl fluoride. The homogenate was centrifuged at 15,000g for 30 min, the supernatant was dialyzed in 15 mM Tris (pH 7.2) containing 5 mM 
