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Agronomy Journal 93:196-206 (2001)
© 2001 American Society of Agronomy

WHEAT

Water Deficit–Induced Senescence and Its Relationship to the Remobilization of Pre-Stored Carbon in Wheat during Grain Filling

Jianchang Yang, Jianhua Zhang, Zhiqing Wang, Qingsen Zhu and Lijun Liu

Dep. of Biology, Hong Kong Baptist Univ., Kowloon Tong, Hong Kong, China

Corresponding author (jzhang{at}net1.hkbu.edu.hk)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 
Remobilization and transfer of the pre-stored food in vegetative tissues to the grains in monocarpic plants require the initiation of whole plant senescence. However, mechanisms by which plant senescence promotes remobilization of assimilates are rather obscure. This study examined the relationship between the senescence induced by water deficits and C remobilization during grain fill. Two semi-winter wheat cultivars (Triticum aestivum L.),Yangmai 158 and Yangmai 931, were treated with two levels of nitrogen (normal [NN] or high [HN]) and three levels of soil moisture (well-watered, moderate water deficit, and severe water deficit). Results showed that water deficits enhanced the senescence by accelerating loss of leaf nitrogen and chlorophyll and increasing lipid peroxidation. At maturity, 75 to 92% of pre-anthesis 14C stored in the straw was reallocated to grains in water-deficit treatments, 50 to 80% higher than the amount in well-watered treatments, indicating that water deficits promoted remobilization. The peak values of abscisic acid (ABA) in both leaves and grains under water-deficit treatments were 63 to 144% higher than those under well-watered treatments. The elevated ABA level correlated with the degree of earlier leaf senescence, the 14C partitioning into grains, and the carbon remobilization. The activites of both acid invertase (INV) and sucrose synthase (SS) in grains were also enhanced by water deficits at the midstage of grain fill. Our results suggest that the senescence and remobilization promoted by water deficits during grain fill are coupled processes in wheat, and elevated ABA concentration may play a regulative role.

Abbreviations: ABA, abscisic acid • DAA, days after anthesis • DAS, days after sowing • ELISA, enzyme-linked immunosorbent assay • HN, high amount of nitrogen • INV, acid invertase • MDA, malondialdehyde • NN, normal amount of nitrogen • NSC, nonstructural carbohydrate • PBS, phosphate buffer saline • {psi}leaf, leaf water potential • {psi}soil, soil water potential • SS, sucrose synthase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results
 Discussion
 REFERENCES
 
WHOLE plant senescence in monocarpic plants such as wheat is the final stage in growth and development (Okatan et al., 1981; Nooden, 1988a). It is an active, ordered process that involves remobilization of stored food from stems and sheaths to the grains (Gan and Amasino, 1997; Nooden et al., 1997; Ori et al., 1999). Delayed senescence retards remobilization and can lead to reduced grain weight and much nonstructural carbohydrate (NSC) left in the straw when growing seasons are terminated (Yang et al., 1997; Zhu et al., 1997). Extensive studies have demonstrated that post-anthesis water deficits result in early senescence and more remobilization of pre-anthesis stored assimilates to grains in cereals (Gallagher et al., 1976; Johnson and Moss, 1976; Jones and Rawson, 1979; Sharp and Davies, 1979; Austin et al., 1980; Lauer and Simmons, 1985; Nicolas et al., 1985; Kobata et al., 1992; Palta et al., 1994; Zhang et al., 1998). However, mechanisms by which plant senescence promotes remobilization of assimilates are rather obscure. Very little is known about the relationship between these two processes, a surprisingly neglected aspect given its importance to yield formation.

Many factors contribute to plant senescence (Nooden, 1988b; Smart, 1994; Lers et al., 1998), but ABA is considered the most promising of the senescence promoters (Lindoo and Nooden, 1978; Biswas and Choudhuri, 1980; Ronen and Mayak, 1981; Nooden, 1988a; Madhu et al., 1999; Tadas et al., 1999). Abscisic acid also has been reported to be an important regulator in transporting assimilates to the developing seeds or fruits (Dewdney and McWha, 1978, 1979; Tietz et al., 1981; Dorffling et al., 1984; Clifford et al., 1986; Brenner and Cheikh, 1995; Yang et al., 1999). However, data on ABA involvement in regulating both senescence and assimilate remobilization have been controversial (Wagner, 1974; Setter et al., 1980; Nooden, 1988a; Barratt et al., 1989; Ober and Setter, 1990; Brown et al., 1991; de Bruijn and Vreugdenhil, 1992).

The objective of this study was to investigate senescence and re-translocation of carbon in wheat subjected to water deficits at the mid and late grain filling stages. The changing pattern of ABA during water deficits and its relationship to the two processes also were investigated.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results
 Discussion
 REFERENCES
 
Plant Materials
The experiment was conducted at Yangzhou University, Yangzhou, China (32°30' N, 119°25' E) from November 1998 to June 1999. Two semi-winter cultivars (Yangmai 158 and Yangmai 931) currently used in local production were grown in porcelain pots. Each porcelain pot (30-cm height, 25-cm diameter, 14.72-L volume) was filled with 18 kg sandy loam soil (Typic fluvaquents, Entisols [U.S. taxonomy]) that contained organic matter at 2.45% and available N–P–K at 105, 33.5, and 66.0 mg kg-1, respectively. On the day of sowing (10 November), 1 g N as urea and 0.2 g P as single superphosphate were mixed into the soil in each pot. At 30 and 112 days after sowing (DAS), 0.4 g N and 0.5 g N as urea were topdressed into each pot, respectively. The pots were placed in a field and sheltered from rain by a removable polyethylene shelter.

Sixteen seeds were sown in each pot. At the three-leaf stage, plants were thinned to eight plants per pot (equivalent to a density of 163 plants m-2). The plants were watered daily by hand to maintain a soil water content close to field capacity (soil water potential, {psi}soil, at -0.01 to -0.02 MPa) until 9 DAA, when water-deficit treatments were initiated. Yangmai 158 and Yangmai 931 headed 154 DAS and 155 DAS, respectively, and flowered at 161 to 167 DAS. At the initial heading (10% of plants headed), the total number of spikes was adjusted to 36 in each pot by removing one to three unproductive tillers from the pot to minimize possible nontreatment effects. The average daily temperatures from anthesis to harvest (161–204 DAS) were 15.3, 17.1, 20.7, 21.2, and 24.6°C, respectively, for each 10-d period.

Nitrogen and Soil Drying Treatments
The experiment was a 2 x 2 x 3 (two cultivars, two levels of nitrogen, and three levels of soil moisture) factorial design with 12 treatment combinations. Each treatment was replicated 30 times in individual pots, of which 24 were used for destructive sampling during the experiment and six for harvest. When the main stem of wheat in pots was at 50% heading (154 DAS for Yangmai 158 and 155 DAS for Yangmai 931), two levels of nitrogen treatments were applied. One-half of the pots were topdressed with either 0.5 g N (normal amount, NN) or 1.2 g N (high amount, HN) as urea per pot. From 9 DAA (163 DAS for both cultivars) to maturity, three levels of {psi}soil were imposed on the plants of both the NN and HN treatments. The well-watered treatment was maintained at -0.02 MPa, the moderate water-deficit treatment was maintained at -0.04 MPa, and the severe water-deficit treatment was maintained at -0.06 MPa. Soil water potential was monitored in the 15- to 20-cm soil depth. A tension meter consisting of a 5-cm-long sensor was installed in each pot to monitor. Tension meter readings were recorded every 2 h from 0600 to 1800 h. When the reading dropped to the designed value, 100, 80, and 60 mL tap water per pot was added to the well-watered, moderate water-deficit, and severe water-deficit treatments, respectively.

Radioactive Labeling
At the boot stage (143 DAS for Yangmai 158 and 144 DAS for Yangmai 931), 40 main stems from each treatment were labeled with 14CO2. Flag leaves were used for labeling between 0900 and 1100 h on a clear day with photosynthetically active radiation at the top of the canopy ranging between 900 and 1100 µmol m-2 s-1. The whole flag leaf was placed into a polyethylene chamber (25-cm length, 4-cm diameter) and sealed with tape. Six milliliters of air in the chamber was drawn out and the same volume of mixed gas containing 14CO2 was injected into the chamber (1% CO2 concentration at specific radioactivity of 14C at 1.48 MBq L-1). The chamber was removed after 30 min.

Labeled plants were harvested at 0 (at 50% anthesis), 9 (the initiation of water withholding), 15, 21, and 27 DAA and at maturity, respectively. Plants from six pots were harvested from each treatment. Harvested plants were divided into leaves, stems plus sheaths, and spikes. Samples were dried in an oven at 80°C to constant weight, ground into powder, and then extracted by shaking in 80% boiling ethanol. The radioactivity of 14C in the extracted aliquots was counted using a liquid scintillation counter (Beckman Instruments, Fullerton, CA) (see Ge et al., 1996). Radioactivity distribution to each part of the plant was expressed as a percentage of total radioactivity remaining in the aboveground portion of the plant.

Sampling and Measurement
One-hundred twenty spikes that headed on the same day were chosen and tagged for each treatment. Twelve to fifteen tagged plants from each treatment were sampled every 3 d from 9 to 27 DAA. The flag leaves and the second and third grains from each spikelet were frozen in liquid nitrogen for 1 min and then stored at -72°C for ABA assay and enzymatic measurement. Aboveground biomass was measured at anthesis and grain maturity. At each harvest, 40 to 50 plants were taken from each treatment and divided into leaves, stems and sheaths, and spikes. All plant parts were dried to constant weight and weighed. Contents of chlorophyll, nitrogen, and malondialdehyde (MDA) in the flag leaves, and soluble sugars and starch in the stem and sheath were measured at 9, 15, 21, and 27 DAA and at maturity, respectively. Each measurement was done on plants from six different pots.

Leaf Water Potential ({psi}leaf)
The measurement of {psi}leaf was made at midday (1200 h) during the first week from water withholding (9–16 DAA). Well-illuminated flag leaves were chosen randomly for these measurements. A pressure chamber (Model 3000, Soil Moisture Equipment Corp., Santa Barbara, CA) was used with six leaves for each treatment.

Abscisic Acid Extraction, Purification, and Quantification
The method for extraction and purification of ABA was modified from that described by Bollmark et al. (1988) and He (1993)(p. 60–68). Samples of 2 to 3 g of frozen flag leaves or grains were ground in a mortar (at 0°C) in 10 mL 80% (v/v) methanol extraction medium containing 1 mmol L-1 butylated hydroxytoluene as an antioxidant. The extract was incubated at 4°C for 4 h and centrifuged at 4000 rpm for 15 min, also at 4°C. The supernatant was passed through a C18 SepPark column (Waters Assoc., Bedford, MA), which had been prewashed with 10 mL 100% and 5 mL 80% methanol, respectively. The column was then washed and eluted with 10 mL 100% methanol and 10 mL ether, respectively. The hormone fraction eluted with methanol and ether from the column was dried under N2. It was then dissolved in 2 mL phosphate buffer saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5) for analysis by an enzyme-linked immunosorbent assay (ELISA).

The antigen and antibody against ABA and the immunoglobulin G-horse radish peroxidase (Ig G-HRP) used in ELISA were produced at the Phytohormones Research Institute, China Agricultural University, China (see He, 1993, p. 60–68). The ELISA was performed on a 96-well microtitration plate. Each well on the plate was coated with a 100-µL coating buffer (1.5 g L-1 Na2CO3, 2.93 g L-1 NaHCO3, 0.02 g L-1 NaN3; pH 9.6) containing 0.25 mg L-1 rabbit antigen against ABA, incubated for 4 h at 37°C, and then kept at room temperature for 30 to 40 min. After washing four times with PBS + Tween 20 (0.1%, v/v) buffer (pH 7.4), each well was filled with 50 µL of either extract or ABA standards (0–2000 µg L-1 dilution range), and 50 µL of 20 mg L-1 anti-(±)ABA antibody against ABA. The plate was incubated for 3 h at 28°C, and then washed as above. To each well 100 µL of 1.25 mg L-1 Ig G-HRP substrate was added and incubated for 1 h at 30°C. The plate was rinsed five times with PBS + Tween 20 buffer, and 100 µL of color-appearing solution containing 1.5 g L-1 0-phenylenediamine and 0.008% (v/v) H2O2 was added to each well. The reaction was stopped by adding of 50 µL 3 M H2SO4 per well when the 2000 µg mL-1 standard had a pale color and the 0 ng µL-1 standard had a deep color in the wells. Color development in each well was detected using an ELISA reader (Model EL310, Bio-TEK Instruments, Winooski, VT) at OD490 nm absorbance. Abscisic acid content was calculated after Weiler et al. (1981). The results are the mean ± SE of at least four replicate incubations. Recoveries of ABA in leaves and in grains were 77.59 ± 4.58 and 79.35 ± 5.64, respectively.

Enzyme Extraction and Assays
The method for INV (EC 3.2.1.26) and SS (EC 2.4.1.13) extraction was modified from Ranwala and Miller (1998). All operations were carried out on ice or at 0 to 4°C. For INV extraction, 1 g fresh leaves or 1 g fresh grains was homogenized in a chilled mortar with 4 mL of chilled 100 mM Tris buffer (pH 7.2) containing 5 mM ß-mercaptoethanaol, 10 mM isoascorbate, and 1 mM phenylmethyl-sulfonyl fluoride (PMSF). The homogenate was centrifuged at 20000 rpm for 30 min. The supernatant was dialyzed overnight in 15 mM Tris (pH 7.2) containing 5 mM ß-mercaptoethanaol, and the dialyzate was used for INV assay.

For SS extraction, leaves or grains were homogenized with a mortar and pestle (5 mL buffer per g fresh weight) 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 20000 rpm for 30 min, the supernatant was desalted on a Sephadex G-25 column (Pharmacia, Freiburg, Germany) 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.

Sucrose synthase was assayed in the cleavage direction. Both INV and SS were analyzed as described by Ranwala and Miller (1998). Enzyme activities were expressed as mol sucrose hydrolyzed per min per kg protein. Protein content was determined according to Bradford (1976), using bovine serum albumin as a standard.

Malondialdehyde content was determined according to Heath and Packer (1968). Soluble sugars and starch contents were determined according to Gong and Zhang (1995)(p. 30–45). Chlorophyll content was determined according to Holden (1976). Nitrogen content was determined by micro-Kjeldahl digestion and distillation, as described by Makino et al. (1983).

Exogenous Abscisic Acid Application
An extra 30 pots of plants with high N–well-watered treatments for each cultivar were arranged for exogenous ABA application. We sprayed 5 x 10-5 M ABA on the leaves at 9 DAA (50 mL per pot). The same volume of distilled water was sprayed on leaves as a control. Abscisic acid in both leaves and grains and INV and SS activities in grains were measured with the methods described above on 3 and 9 d after ABA application (12 and 18 DAA). Five pots of plants for each treatment were harvested for examination of grain weight and carbon reserve in stems and sheaths. Each measurement had 5 replicates.

The results were analyzed using analysis of variance (ANOVA). Data from each sampling date were analyzed separately. Means were tested by least significant difference at the P0.05 level (LSD0.05). The differences of the data between the two cultivars were not significant statistically but they are still presented separately for clarity.

This experiment was also conducted under field conditions at the same time the pot experiment was conducted. The results of the pot and field experiments were very similar. The pot experiment only was reported in this paper because of limited space.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
Discussion
 REFERENCES
 
Leaf Water Potential
Midday {psi}leaf during the first week after water withholding is shown in Fig. 1 . The {psi}leaf for well-watered plants was about -0.9 to -1.1 MPa (Fig. 1). Controlled watering decreased the {psi}leaf. The {psi}leaf reached -1.3 to -1.6 MPa on the third day under the moderate water-deficit treatment and -1.8 to -2.0 MPa on the fourth day under the severe water-deficit treatment. Under the same water-deficit level, HN plants had a slightly lower {psi}leaf than the NN plants, suggesting that the leaves with high nitrogen nutrition might lose more water.



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  		Fig. 1 Changes of leaf water potentials of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) during the first week when soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). Measurements were made on the flag leaves at midday (1200 h). Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
Senescence Parameters
Nitrogen and chlorophyll contents were monitored as an objective way to quantify senescence. Both nitrogen and chlorophyll contents (Fig. 2) in the flag leaf for well-watered plants gradually declined during grain filling. Regression coefficients (slopes) of chlorophyll concentrations (Y1) to measurement date (X) were -0.477 to -0.858, and slopes of nitrogen contents (Y2) to X were -0.072 to -0.115, respectively, under well-watered treatments. The rate of decline was increased after plants were exposed to water deficits. Slopes of Y1 to X and Y2 to X were -1.198 to -1.197 and -0.149 to -0.278, respectively, under water deficits. However, plants with HN maintained higher nitrogen and chlorophyll contents in leaves relative to those with NN at the same water-deficit level.



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  		Fig. 2 Changes of nitrogen and chlorophyll content in the flag leaves of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) after soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). Arrows in the figures indicate the start of soil drying treatments. Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
In contrast to the decrease in nitrogen and chlorophyll contents, the lipid peroxidation (indicated by MDA content) in flag leaves was higher in plants under water deficits (Fig. 3) . The more severe the water deficit, the higher the MDA in leaves. Plants with HN had lower lipid peroxidation when compared with those with NN when subjected to the same level of water deficit. The reduced nitrogen and chlorophyll concentrations in association with the increased lipid peroxidation in the leaves under moderate and severe water-deficit treatments indicated that water deficits promoted plant senescence, while high nitrogen delayed this process.



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  		Fig. 3 Changes of lipid peroxidation, shown as malondialdehyde (MDA) content, in the flag leaves of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) after soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). Arrows in the figures indicate the start of soil drying treatments. Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
Remobilization of Carbon Reserve
Figure 4 shows the disappearance of pre-anthesis assimilates (14C) in the stem and sheath and appearance in grains during grain filling. At the start of water withholding (9 DAA), about 75% of 14C fed to the flag leaves at the boot stage was partitioned in the stem and sheath, and about 10% in the grains. After 12 d (21 DAA), 14C in the stem and sheath was reduced to 25 to 31% under moderate water-deficit, 13 to 19% under severe water-deficit, and 51 to 57% under well-watered treatments at the NN level. Opposite to that observed in the stem and sheath, the 14C level in grains increased by 51 to 60% under moderate water-deficit, 70 to 72% under severe water-deficit, and only 20 to 32% under well-watered treatments at 21 DAA. At maturity, 75 to 92% of pre-anthesis 14C stored in the stem and sheath was reallocated to grains under water-deficit treatments, 50 to 80% higher than the amount under well-watered treatments. Compared with NN, HN reduced reallocation of 14C from the stem and sheath to grains when {psi}soil was the same.



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  		Fig. 4 Changes of 14C partitioning in the stem plus sheath and grains of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) after soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). The 14CO2 was fed to the flag leaves at booting stage. Arrows in the figures indicate the start of soil drying treatments. Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
Very similar to 14C reallocation, soluble sugars and starch in the stem and sheath were decreased under water deficits, resulting in a remarkable reduction in total sugars (NSC) (Fig. 5) . However, reduction in starch under water-deficit treatments was more remarkable than that in soluble sugars if compared with well-watered treatments, which indicated that more starch was hydrolyzed in the stem and sheath when subjected to water deficits.



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  		Fig. 5 Changes of nonstructural carbohydrate and soluble sugars plus starch in the stem and sheath of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) after soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). Arrows in the figures indicate the start of soil drying treatments. Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
Kernel Weight and Grain Yield
Kernel weight was reduced by water deficits under NN, but increased under HN when compared with respective well-watered treatments (Table 1). A similar result was obtained for grain yield (Table 1), possibly because only the kernel weight, rather than the spike number or kernel number per spike, was influenced by water deficits during grain filling.


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  		Table 1 Kernel weight and grain yield of wheat subjected to various N and soil moisture treatments (refer to Fig. 1). Values are means of 216 plants harvested from six pots of each treatment

 
Abscisic Acid in Leaves and Grains
Abscisic acid accumulation in leaves of plants under water deficits was greater than that under well-watered treatments (Fig. 6) . However, the accumulation pattern of ABA in leaves varied with the treatments. Under well-watered treatments, both at NN and HN, ABA in leaves gradually increased during grain filling. Under moderate and severe water deficits, ABA in the leaves increased dramatically during the first 6 d (9–15 DAA) at NN, or during the first 9 d (9–18 DAA) at HN, after water was withheld. Thereafter, ABA decreased in these treatments. Plants under HN had lower ABA in leaves than those under NN when subjected to same level of soil moisture. The pattern of ABA accumulation in the grains (Fig. 6) was similar to that in the leaves.



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  		Fig. 6 Changes of abscisic acid (ABA) contents in the flag leaves and grains of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) after soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). Soil drying treatments were started 9 d after anthesis. Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
The peak values of ABA in both leaves and grains were correlated to leaf senescence, that is, the higher the peak value of ABA content, the more nitrogen and chlorophyll lost or more lipid peroxidation increased in leaves. This suggests that ABA may initiate or regulate plant senescence (Fig. 2, 3, and 6).

Acid Invertase and Sucrose Synthase Activities in Grains
For all the treatments, INV and SS activities in grains increased initially and declined after reaching a maximum. However, the peak values and the time to reach the peak were different among the treatments. In general, severe water-deficit treatments had the highest peak, followed by moderate water-deficit treatment peaks, at either NN or HN. The activities peaked earlier at NN than at HN (Fig. 7) .



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  		Fig. 7 Changes of activities of acid invertase (INV) and sucrose synthase (SS) in grains of the semi-winter wheat cultivars Yangmai 158 (A) and Yangmai 931 (B) after soil drying was applied. NN and HN indicate normal and high levels of nitrogen application at heading time, respectively. WW, MD, and SD are well-watered, mildly dried, and severely dried treatments at grain filling, respectively (see details in the Materials and Methods section). Arrows in the figures indicate the start of soil drying treatments. Vertical bars represent ±SE of the mean where these exceed the size of the symbol

 
Abscisic acid accumulation reached a maximum earlier than INV and SS activities (refer to Fig. 6 and 7). The maximum values of both enzyme activities significantly correlated with that of ABA accumulation in both leaves and grains (Fig. 8) , suggesting that INV and SS activities in grains might be regulated by the ABA accumulation in the plants.



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  		Fig. 8 Relationship of abscisic acid (ABA) in leaves and grains with the acid invertase (INV) and synthase sucrose (SS) activities during grain filling in the semi-winter wheat cultivars Yangmai 158 (•) and Yangmai 931 ({circ}). Data represent the peak values of each treatment during water withholding. Correlation coefficients (r) are calculated with stars indicating statistical significance at the P0.05 (*) or P0.01 (**) levels

 
Sucrose synthase activity in grains was a little higher and its maximum was earlier than INV. However, activity of INV was higher in plants under water deficits than that of SS before its decline.

Correlation between Abscisic Acid, Acid Invertase, Sucrose Synthase, and the Remobilization of Pre-Stored Carbon
The pre-fixed 14C disappearance in the stem and sheath (14C at anthesis minus 14C at maturity), the 14C increase in grains (14C at maturity minus 14C at anthesis), and the total remobilized carbon reserve in the stem and sheath ([NSC in stems and sheaths at anthesis (a) - NSC residue at maturity]/a x 100) were calculated as remobilization parameters. The correlations of the peak value of ABA either in leaves or in grains during grain filling with remobilization parameters, and of the peak value of either INV or SS activities with the parameters were determined. All the correlation coefficients between these measurements were positive and significant at high probability levels (Table 2).


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  		Table 2 Correlations of abscisic acid (ABA) concentrations in leaves and grains and activities of acid invertase (INV) and sucrose synthase (SS) in grains with the pre-fixed 14C disappearance in the stem and sheath (14C at anthesis - 14C at maturity), 14C increase in grains (14C at maturity - 14C at anthesis), and remobilized carbon reserve in the stem and sheath ([nonstructural carbohydrate in stems and sheaths at anthesis (a) - nonstructural carbohydrate residue at maturity]/a x 100). Data used for the calculations are from Fig. 4, 5, 6, and 7

 
Effect of Exogenous Abscisic Acid Application
When ABA was applied to NN and well-watered plants at early grain filling stage (9 DAA), ABA in both leaves and grains and activities of INV and SS in grains were significantly increased. The remobilized carbon reserve in the stem and sheath was doubled and grain weight increased by 16.37 to 22.17% in the ABA treatment compared with that of the control (Table 3).


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  		Table 3 Effect of exogenously applied abscisic acid (ABA) on acid invertase (INV) and sucrose synthase (SS) activities, remobilization of carbon, and grain weight of the plants grown in well-watered pots with a high amount of nitrogen applied. We sprayed 5 x 10-5 M ABA on the leaves at 9 d after anthesis (see Materials and Methods section). Each measurement was five replications

 

   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
REFERENCES
 
We found that when soil drying was controlled during the mid and late grain filling period, as indicated by the moderate leaf water deficits (Fig. 1), early plant senescence was induced (Fig. 2 and 3). This induced early senescence promoted the remobilization of pre-anthesis carbon stored in the stem and sheath to the grains (Fig. 4 and 5). The more quickly and severe the senescence, the more rapidly more carbon was reallocated from stems and sheaths to grains. The kernel weight and grain yield were increased under water deficits when a high amount of nitrogen was applied (Table 1). We therefore conclude that the senescence and the remobilization apparently promoted by water deficits during the grain filling period are coupled processes in wheat. This may have importance in maintaining grain yield against water deficits during the grain filling period (Kobata et al., 1992; Palta et al., 1994; Gebbing and Schnyder, 1999), and would be beneficial in years when a hot and dry wind (temperature > 30°C, relative humidity < 30%, and wind speed > 3 m s-1) dehydrates the wheat before its maturity, or in cases where senescence is unfavorably delayed by heavy nitrogen use (Zhang et al., 1998).

Our earlier work has shown that xylem ABA concentration is sensitive to water deficits and is mainly responsible for the physiological regulation of shoots (Zhang and Davies, 1989, 1990a,b; Davies and Zhang, 1991; Tardieu et al., 1993). In the present study we found that ABA in both leaves and grains greatly increased under water-deficit treatments (Fig. 6). The extent to which ABA was elevated (the peak value) was correlated with the degree to which leaves senesced (Fig. 2 and 3). We speculate that water deficit–induced senescence is related to or regulated by the elevated ABA concentrations in the plants.

It is believed that two major enzymes, INV and SS, are involved in the unloading of assimilates in the sink through cleavage of the sucrose (the transported form of carbon assimilates in plants) to its constituent monosaccharides (glucose and fructose), which are used either in metabolic or biosynthetic reactions (Weber et al., 1995; Zrenner et al., 1995; Ranwala and Miller, 1998). Our results show that the activities of both INV and SS in grains were higher under water deficits (Fig. 7), and their peak values were positively and closely correlated with 14C partitioning into grains, and with total remobilized carbon reserve in the stem and sheath (Table 2). We conclude that the enhanced activities of the two enzymes in the grains contributed to the increased remobilization of assimilates to grains under water deficits.

It is notable that the changing patterns of INV and SS activities (Fig. 7) are associated with that of ABA (Fig. 6). The times taken to reach peak activity were similar, and a positive and significant correlation between ABA content and the enzyme activities was found (Fig. 8). Furthermore, the activities of both the enzymes and the remobilization were significantly increased when exogenous ABA was applied at the early grain filling stage (Table 3). The results suggest that the senescence and remobilization promoted by water deficits during the grain filling period are coupled processes in wheat, and the elevated ABA concentration may play a regulative role. Obviously further investigation is needed to establish a possible cause and effect relationship.


   ACKNOWLEDGMENTS
 
We are grateful for grants from the Faculty Research Grant of Hong Kong Baptist University, the Area of Excellence Research Fund of the Chinese University of Hong Kong, and the Research Grants Council of the Hong Kong University Grants Committee.

Received for publication February 11, 2000.
   REFERENCES
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 ABSTRACT
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