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Keywords:

  • Triticum aestivum;
  • abscisic acid;
  • carbon remobilization;
  • cytokinins;
  • grain filling;
  • nitrogen;
  • senescence;
  • water stress;
  • zeatin;
  • zeatin riboside

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This study investigated the possibility that abscisic acid (ABA) and cytokinins may mediate the effect of water deficit that enhances plant senescence and remobilization of pre-stored carbon reserves. Two high lodging-resistant wheat (Triticum aestivum L.) cultivars were field grown and treated with either a normal or high amount of nitrogen at heading. Well-watered (WW) and water-stressed (WS) treatments were imposed from 9 d post-anthesis until maturity. Chlorophyll (Chl) and photosynthetic rate (Pr) of the flag leaves declined faster in WS plants than in WW plants, indicating that the water deficit enhanced senescence. Water stress facilitated the reduction of non-structural carbohydrate in the stems and promoted the re-allocation of prefixed 14C from the stems to grains, shortened the grain filling period and increased the grain filling rate. Water stress substantially increased ABA but reduced zeatin (Z) + zeatin riboside (ZR) concentrations in the stems and leaves. ABA correlated significantly and negatively, whereas Z + ZR correlated positively, with Pr and Chl of the flag leaves. ABA but not Z + ZR, was positively and significantly correlated with remobilization of pre-stored carbon and grain filling rate. Exogenous ABA reduced Chl in the flag leaves, enhanced the remobilization, and increased grain filling rate. Spraying with kinetin had the opposite effect. The results suggest that both ABA and cytokinins are involved in controlling plant senescence, and an enhanced carbon remobilization and accelerated grain filling rate are attributed to an elevated ABA level in wheat plants when subjected to water stress.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Whole plant senescence in monocarpic plants such as wheat (Triticum aestivum L.) is the final stage in growth and development (Okatan, Kahanak & Nooden 1981; Nooden 1988a; Nooden, Guiamet & John 1997). It is a genetically programmed process that involves remobilization of nutrients from vegetative tissues to grains (Buchanan-Wollaston 1997; Gan & Amasino 1997; Nooden et al. 1997; Ori et al. 1999). Delayed senescence, which in practice is induced by either too much nitrogen fertilizer or an adoption of lodging-resistant cultivars that stay ‘green’ for too long or plants that remain green when the grains are due to ripe, retards such remobilization and can lead to slow grain filling and low harvest index (Peng, Guo & Yan 1992; Zhang et al. 1998). Our early work (Yang et al. 2000a, 2001a) has shown that water stress during the grain filling of wheat and rice (Oryza sativa L.) can enhance plant senescence and lead to faster and better remobilization of pre-stored carbon from vegetative tissues to the grains and an acceleration of the grain filling rate. However, the mechanisms by which plant senescence promotes remobilization of assimilates remain unclear, and very little is known about the relationship between these two processes.

Many factors, either endogenous or environmental, contribute to plant senescence (Nooden 1988b; Smart 1994; Lers et al. 1998; Pic et al. 2002), but abscisic acid (ABA) and cytokinins are generally believed to be two major regulators of plant senescence (Biswas & Choudhuri 1980; Nooden 1988a; Zeevaart & Creelman 1988; Haberer & Kieber 2002). High levels of cytokinins in plants can delay senescence (Buchanan-Wollaston 1997; Nooden et al. 1997), whereas a high ABA concentration has opposing effects (Nooden 1988b; Madhu, Thomas & Edward 1999; Tadas et al. 1999). Both ABA and cytokinins are also considered as key regulators to seed development (Davies 1987; Brenner & Cheikh 1995). In barley (Hordeum vulgare L.), maize (Zea mays L.), rice and wheat, high levels of cytokinins are generally found in the endosperm of developing seeds, which may be required for the cell division during the early phase of seed setting (Michael & Seiler-Kelbitsch 1972; Saha, Nagar & Sircar 1986; Morris et al. 1993; Dietrich et al. 1995; Banowetz, Ammar & Chen 1999; Yang et al. 2000b). ABA has been assumed to be an important regulator in transporting assimilates to the developing seeds or fruits (Tietz et al. 1981; Clifford, Offler & Patrick 1986; Brenner & Cheikh 1995). There are numerous reports on the correlation between the level of ABA and the growth rate of fruits or seeds (e.g. Eeuwens & Schwabe 1975; Browning 1980; Berüter 1983; Schussler, Brenner & Brun 1984, 1991; Wang et al. 1987; Ross & McWha 1990; Kato, Sakurai & Kuraishi 1993; Wang et al. 1998; Yang et al. 1999). However, the proposal that ABA is involved in regulating  both  senescence  and  assimilate  mobilization has remained disputable (Jones & Brenner 1987; Nooden 1988b; Barratt et al. 1989; Ober & Setter 1990; Brown, Paliyath & Thompson 1991; Schussler et al. 1991; de Bruijn & Vreugdenhil 1992; Sharp & LeNoble 2002).

The purposes of this study were to investigate the processes of senescence, remobilization of carbon reserves, and grain filling in wheat and changes in ABA and cytokinins (Z + ZR) in the stems, leaves and grains subjected to water stress during grain filling, and determine whether and how the changes of ABA and cytokinins were correlated with these processes.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant materials

The experiment was conducted at Yangzhou University Farm, Yangzhou, China (32°30′ N, 119°25′ E) from November 2001 to June 2002. Two highly lodging-resistant cultivars of semi-winter wheat (Triticum aestivum L.), Yangmai 158 and Yangmai 10, currently used in local production, were grown in the field. The sowing date was 1 November, and the plant density was adjusted to 156 plants m−2 at the three-leaf age. The soil of the field was sandy loam soil [Typic fluvaquents, Entisols (U.S. taxonomy)] that contained organic matter at 2.45% and available N-P-K at 106, 33.8 and 66.4 mg kg−1, respectively. On the day of sowing, 14 g N m−2 as urea and 4 g P m−2 as single superphosphate were applied to the soil. At 30 d after sowing (DAS) and 115 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, ψsoil, at −0.02 to – 0.025 MPa) by manual watering until 9 d post-anthesis (DPA) when water-deficit treatments were initiated. Yangmai 158 and Yangmai 10 headed (50% plants) at 155 and 156 DAS, respectively, and flowered during 162–168 DAS. Air temperatures during grain filling (162–221 DAS), averaged over 10 d, were 15.3, 16.5, 17.8, 19.2, 20.9 and 22.3 °C, respectively.

Nitrogen and water-stress treatments

The experiment was a 2 × 2 × 2 (two cultivars, two levels of N, and two levels of soil moisture) factorial design with eight treatments. Each of the treatment had three plots as repetitions in a complete randomized block design. Plot dimension was in 4 m × 5 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 the plots were top dressed with either 3 g N m−2 (normal amount, NN) or 8 g N m−2 (high amount, HN) as urea. From 9 DPA (174 DAS for both cultivars) to maturity, two levels of ψsoil were imposed on the plants of both NN and HN treatments by controlling water application. The well-watered (WW) treatment was maintained at −0.02 MPa, and the water-stressed (WS) treatment was maintained at −0.08 MPa. Soil water potential was monitored in the 15 to 20 cm soil depth. Five tension meters were installed in each plot to monitor. Tension meter readings were recorded every 6 h from 0600 to 1800 h. When the reading dropped to the designed value, 0.08 and 0.02 m3 of tap water per plot was added to the WW and WS plants, respectively. A rain shelter consisting of a steel frame covered with plastic sheet was used to protect the plot during rain.

Radioactive labelling

At the boot stage (145 DAS for Yangmai 158 and 146 DAS for Yangmai 10), 60 plants from each treatment were labelled with 14CO2. Flag leaves of main stems were used for the labelling between 0900 and 1100 h on a clear day with photosynthetically active radiation at the top of the canopy ranging between 1000 and 1100 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. Six millilitres of air in the chamber was drawn out, and the same volume of gas was injected into the chamber, which contained 10 mol m−3 CO2 at a specific radioactivity of 14C of 1480 MBq m−3. The chamber was removed after 1 h.

Labelled plants were harvested at 0 (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 Inc, Fullerton, CA, USA), 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 above-ground portion of the plant.

Sampling

One hundred and fifty spikes that headed on the same day were chosen and tagged for each plot. Ten tagged plants (leaves + culms + sheaths + spikes) from each plot were sampled at 3 d intervals from anthesis to 21 DPA and 6 d intervals from 27 DPA to maturity. The second and third grains from each spikelet were removed. Half sampled grains, stems (culms + sheaths), and the flag leaves were frozen in liquid nitrogen for 2 min and then stored at −80 °C for hormonal measurement. The other half of the grains, stems, and leaves was used for the measurement of dry weight and non-structural carbohydrate (NSC) in the stems. NSC (soluble sugars and starch) was determined according to the method described earlier (Yang et al. 2000a). The grains for dry weight measurement were dried at 70 °C to constant weight for 72 h, and weighed. The grain-filling process was fitted by Richards's (1959) growth equation as described by Zhu, Cao & Luo (1988):

  • W  =  A/(1  +  Be − kt)1/N(1)

Grain filling rate (G) was calculated as the derivative of Eqn 1:

  • G  =  AKBe − kt/(1  +  Be − kt)(N + 1)/N(2)

where W is the grain weight (mg), A is the final grain weight (mg), t is the time after anthesis (d), and B, k and N are coefficients determined by regression. The active grain filling period was defined as the days when W was from 5% (t1) to 95% (t2) of A. An average grain-filling rate during this period was therefore calculated from t1 to t2.

Grain dry weight per spike was also determined at 9, 18, 27 and 39 DPA, respectively. Twenty spikes from each plot were sampled for each measurement. Plants (except border) from a 4 m2 site from each plot were harvested at maturity for the determination of grain yield. Yield components, namely the spikes per square metre, grain number per spike, and per spike grain weight, were determined from plants harvested from a 1 m2 site (excluding the border plants) randomly sampled from each plot.

Measurement of leaf water potential (ψleaf)

Leaf water potentials of flag leaves were measured at 2 h intervals on 15 and 16 d after withholding water. Well-illuminated flag leaves were chosen randomly for such measurements. A pressure chamber (Model 3000; Soil Moisture Equipment Corp., Santa Barbara, CA, USA) was used for ψleaf measurement with six leaves for each treatment.

Measurement of photosynthetic rate and chlorophyll content

The photosynthetic rate (Pr) of the flag leaves was measured on 0, 9, 15, 20, 27, 33 and 39 DPA. A gas exchange analyser (CID-PS CO2 Analyzer System; CID, Vancouver, WA, USA) was used to measure Pr. Measurements were made during the period 0900–1100 h when photosynthetically active radiation above the canopy was 1000 to 1100 µmol m−2 s−1. Flag leaves were sampled on the above dates for measurement of chlorophyll (Chl) content. The Chl was extracted by shaking in methanol overnight and determined as described by Holden (1976). Six leaves were used for each treatment.

Hormonal extraction, purification and quantification

The methods for extraction and purification of abscisic acid [(+/–) ABA], zeatin (Z), and zeatin riboside (ZR) were modified from those described by Bollmark, Kubat & Eliasson (1988) and He (1993). Samples of 1–3 g of the frozen flag leaves, or stems, or grains were ground in a mortar (at 0 °C) in 10 mL 80% (v/v) methanol extraction medium containing 1 mol m−3 butylated hydroxytoluence as an antioxidant. The extract was incubated at 4 °C for 4 h and centrifuged at 4800 g for 15 min at the same temperature. The supernatants were passed through Chromosep C18 columns (C18 Sep–Pak Cartridge; Waters Corp., Millford, MA, USA), pre-washed with 10 mL 100% and 5 mL 80% methanol, respectively. The hormone fractions were dried under N2, and 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 mouse monoclonal antigens and antibodies against Z, ZR and ABA, and immunoglobulin G–horse radish peroxidase (Ig G–HRP) used in the ELISA were produced at the Phytohormones Research Institute, China Agricultural University, Beijing, China (see He 1993). The method for quantification of ABA, Z and ZR by ELISA has been described previously (Yang et al. 2001b).

Application of ABA and kinetin

Plants were grown in porcelain pots that were placed in a field. Each porcelain pot (30 cm height, 25 cm diameter, 14.72 L volume) was filled with 18 kg sandy loam soil with the same nutrient contents as the field soil. On the day of sowing (1 November), 1 g N as urea and 0.2 g P as single superphosphate were mixed into the soil in each pot. On 30 and 12 DAS and at heading (151–153 DAS), 0.4, 0.5, and 1 g N as urea were top-dressed into each pot, respectively. 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 manually to maintain a soil water content close to field capacity.

Starting at 9 DPA, either 20 × 10−6 m ABA or 40 × 10−6 m kinetin (both from Sigma Chemical Co., St Louis, MO, USA) were sprayed at the rate of 50 mL per pot on the leaves daily for five consecutive days with 0.5% (v/v) Teepol (Fluka, Riedel-de-Haen, Germany) as surfactant. Plants sprayed with the same volume of 0.5% Teepol solution were taken as a control. Each treatment had 60 pots. The Chl content in the flag leaves, grain filling rate, and NSC in stems were measured with the methods described above, with four replications for each measurement. Eight pots of plants from each treatment were harvested at maturity for examination of grain weight.

Statistical analysis

The results were analysed for variance using SAS/STAT statistical analysis package (version 6.12, SAS Institute, Cary, NC, USA). Data from each sampling date were analysed separately. Means were tested by least significant difference at P0.05 level (LSD0.05). Linear regression was used to evaluate the relationship between hormonal concentrations in plant tissues with remobilization of carbon reserves, senescence parameters and the grain filling rate. Since the two cultivars behaved the same, the data are presented as an average between the two cultivars.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Leaf water potential

Figure 1 illustrates the diurnal changes of ψleaf of the flag leaves. The ψleaf ranged from −0.48 MPa at pre-dawn (0600 h) to −1.18 MPa at midday (1200 h) for WW plants. It was greatly reduced during the daytime for WS plants, with a midday ψleaf at −1.67 to −1.83 MPa. The differences in ψleaf between WW and WS plants, either at NN or HN, were very small at pre-dawn and in early morning, indicating that plants subjected to the water stress could rehydrate overnight.

image

Figure 1. Diurnal changes of leaf water potentials of wheat. Measurements were made on the flag leaves at 15 and 16 d after withholding water. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water stressed with high amount of nitrogen (□). Error bars represent ± 1 SE (n = 12).

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Senescence parameters

Photosynthetic rate and Chl content were monitored as an objective way to quantify senescence. As no lodging happened in any treatment, both Chl content and Pr in the flag leaves of WW plants slowly declined with the ageing of leaves (Fig. 2). The WS treatment accelerated such a decline at both NN and HN, suggesting that the water deficit enhanced the plant senescence. As expected, plants with HN maintained a higher Chl content and Pr than with NN at the same level of ψsoil, indicating that HN slowed senescence.

image

Figure 2. Chlorophyll concentration (a) and photosynthetic rate (b) in the flag leaves of wheat. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water-stressed with high amount of nitrogen (□). Arrows in the figure indicate the start of withholding water. Error bars represent ± 1 SE (n = 12).

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Carbon remobilization and grain filling rate

Water stress promoted the re-allocation of pre-anthesis assimilates from the stems to grains. Figure 3 shows the disappearance of pre-anthesis assimilated 14C in the stems and its appearance in 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 stems was reduced to 22–33% for WS plants and to 53–65% for WW plants. At the same time, 14C in grains increased to 38–50% for WS, and only 7–17% for WW plants at 33 DPA. In comparison, HN reduced the 14C re-allocation into the grains (Fig. 3).

image

Figure 3. Changes of 14C partitioning in the stems (a) and grains (b) of wheat. The 14C was fed to the flag leaves at the booting stage. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water stressed with high amount of nitrogen (□). Arrows in the figure indicate the start of withholding water. Error bars represent ± 1 SE (n = 12).

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Similar to the case of 14C re-allocation, the total sugars (soluble sugars + starch) as NSC in the stems of WW plants decreased a little from 9 to 39 DPA but increased thereafter (Fig. 4). The WS treatments accelerated the reduction in NSC in the stems from 9 to 33 DPA. The reduction was faster at NN than at HN when ψsoil was the same. The fast remobilization of carbon reserves under the WS treatment coincided with the fast plant senescence induced by the water deficit shown in Fig. 2.

image

Figure 4. Changes in non-structural carbohydrate (NSC) in the stems of wheat. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water stressed with high amount of nitrogen (□). Arrows in the figure indicate the start of withholding water. Error bars represent ± 1 SE (n = 6).

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At the same level of ψsoil, HN slowed grain filling (Fig. 5). WS treatments increased the grain-filling rate and shortened the grain filling period at both NN and, as shown in Fig. 5, the final grain weight was not significantly different between the WW and WS treatments when NN was applied. However, it was significantly increased under WS–HN treatments. Similar results were obtained with whole grain weight per spike and grain yield (Table 1), because only the grain weight, rather than the spike number or grain number per spike, was significantly influenced by WS and nitrogen treatments during grain filling for this experiment.

image

Figure 5. Grain filling process (a) and grain filling rate (b) of wheat. Grain filling rate was calculated according to Richards (1959) equation. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water stressed with high amount of nitrogen (□). Arrows in the figure indicate the start of withholding water. Error bars in the figure a represent ± 1 SE (n = 6).

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Table 1.  Grain weight per spike (g spike−1), spike number (spike m−2), grain number per spike (grain spike−1), and grain yield (g m−2) of wheat subjected to various N and soil moisture treatments
TreatmentGrain weight per spikeAt maturity
Days post-anthesis
9182739Spike numberGrain numberGrain yield
  1. The treatments are: WW-NN, well watered with normal amount of nitrogen; WS-NN, water stressed with normal amount of nitrogen; WW-HN, well watered with high amount of nitrogen; and WS-HN, water stressed with high amount of nitrogen. Values of grain weight per spike were means of 60 plants harvested from each treatment. Spike number per square metre and grain number per spike were means of the plants from 6 m2 from each treatment. The grain yield was the means of plants harvested from 24 m2 of each treatment. Different letters indicate statistical significance at P= 0.05 level within the same column.

WW-NN0.17 a0.44 b0.93 b1.59 b357 a40.6 a605 ab
WS-NN0.16 a0.64 a1.20 a1.51 b358 a39.7 a576 b
WW-HN0.10 b0.36 c0.73 c1.29 c361 a40.4 a521 c
WS-HN0.11 b0.47 b1.19 a1.71 a356 a40.2 a627 a

Hormonal changes in plant tissues

The methods used in this study for hormone extraction and purification and for quantification of hormones in the leaves, stems, and grains by ELISA recovered 71.1–78.6% of Z, 73.1–81.1% of ZR, and 75.3–82% of ABA (Table 2). The specificity of the monoclonal antibodies and the other possible non-specific immunoreactive interference were checked previously and proved reliable (Wu, Chen & Zhou 1988; Zhang, He & Wu 1991; Yang et al. 2002).

Table 2.  Recovery test of enzyme-linked immunosorbent assay (ELISA) for zeatin (Z), zeatin riboside (ZR), and abscisic acid (ABA). The test wheat cultivar was Yangmai 158
Plant organHormonePlant sampleaPant sample + standardbPercentage recovery
  1. Data are expressed as means ± standard error (SE; n= 6). aGrains only (pmol g−1 FW). bGrains + synthetic compounds (pmol g−1 FW) of Z, ZR, and ABA, respectively. Two hundred picomole of each compound was added to 1 g fresh plant sample before purification. The compounds were from Sigma Chemical Co.

LeavesZ 5.6 ± 0.4160 ± 14.277.8 ± 6.3
ZR 7.8 ± 0.6165 ± 15.579.4 ± 7.1
ABA 163 ± 12.4291 ± 20.780.2 ± 6.8
StemsZ12.4 ± 1.1151 ± 13.271.1 ± 7.3
ZR17.6 ± 1.5159 ± 14.173.1 ± 6.9
ABA64.2 ± 5.8199 ± 16.775.3 ± 6.4
GrainsZ58.3 ± 4.7203 ± 17.478.6 ± 6.7
ZR83.5 ± 7.2230 ± 20.681.1 ± 7.5
ABA 212 ± 17.4338 ± 30.782.0 ± 6.9

The concentration of ZR in either the flag leaves, or stems, or grains was 40–43% greater than that of Z (Table 2), and both showed a similar changing pattern during grain filling (data not shown). The Z + ZR concentrations in the flag leaves of WW plants slowly declined with the ageing of leaves (Fig. 6a). Water stress accelerated such a decline at both NN and HN. The concentration of Z + ZR was higher at HN than at NN although ψsoil was the same, in good agreement with Chl content changes in the leaves (refer to Fig. 2).

image

Figure 6. Changes in zeatin + zeatin riboside concentrations in the flag leaves (a), stems (b), and grains (c) of wheat. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water stressed with high amount of nitrogen (□). Arrows in the figure indicate the start of withholding water. Error bars represent ± 1 SE (n = 6).

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The Z + ZR concentrations in the stems and grains showed a similar changing pattern (Fig. 6b & c). They sharply increased during the early grain filling stage, reached a maximum at 6 DPA under the NN and 9 DPA under HN treatments, and dropped very quickly thereafter. During the first week of withholding water (9–15 DPA), the difference in Z + ZR concentrations was not significant between WW and WS plants. WW plants had higher Z + ZR concentrations in the stems and grains than WS plants only at the mid and late grain filling stages (18 DPA afterwards). The peak values of Z + ZR concentrations were lower at HN than at NN in both the stems and grains.

In contrast to the appearance of Z + ZR, ABA concentrations in the leaves and stems for WW plants were rather low and little changed during grain filling, but they were substantially enhanced by WS treatments (Fig. 7). Elevated ABA levels in WS stems were associated with the increase of 14C in the grains under WS treatments as shown in Fig. 3b.

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Figure 7. Changes in abscisic acid concentrations in the flag leaves (a), stems (b), and grains (c) of wheat. The treatments are: well watered with normal amount of nitrogen (•), water stressed with normal amount of nitrogen (○), well watered with high amount of nitrogen (▪), and water stressed with high amount of nitrogen (□). Arrows in the figure indicate the start of withholding water. Error bars represent ± 1 SE (n = 6).

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ABA concentrations in the grains were very low but they slowly increased at the early grain filling stage, and reached a maximum at 27 DPA for WW plants at both NN and HN (Fig. 7c). The ABA level in the grains was remarkably enhanced by water stress and reached a maximum at 18 DPA at NN and 21 DPA at HN. At the same level of ψsoil, plants with HN contained less ABA in the tissues than those with NN. Changes of ABA concentrations in the grains were paralleled by the grain filling rate (refer to Fig. 5b).

Correlations of hormones with remobilization and grain filling rate

The disappearance of NSC in the stems (NSC at anthesis minus NSC at 9, 15, 21, 27 and 33 DPA, respectively) and 14C transfer to the grains (14C at 9, 15, 21, 27 and 33 DPA, respectively, minus 14C at anthesis) were calculated as remobilization parameters, The correlations of ABA and Z + ZR concentrations in the leaves and stems during a rapid NSC remobilization in the stems (9–33 DPA) with the remobilization parameters, Pr and Chl content, in the flag leaves were determined (Table 3). The ABA concentration in both the leaves and stems was significantly and positively correlated with NSC remobilization in the stems and 14C increase in grains (r = 0.83** to 0.94**, P = 0.01), and negatively and significantly correlated with the Pr and Chl content (r = −0.83** to −0.92**, P = 0.01). In contrast to ABA, the concentration of Z + ZR was positively and significantly correlated with the Pr and Chl content (r = 0.69** to 0.97**, P = 0.01), and negatively and significantly correlated with the remobilization parameters (r = −0.81** to −0.89**, P = 0.01) (Table 3).

Table 3.  Correlation coefficients of abscisic acid (ABA) and zeatin (Z) + zeatin riboside (ZR) concentrations in the stems and flag leaves with non-structural carbohydrate (NSC) mobilization in the stem (NSC at anthesis minus NSC at 9, 15, 21, 27 and 33 d post-anthesis, respectively), 14C partitioning in grains, photosynthetic rate and chlorophyll content of the flag leaves during grain filling of wheat
Correlation with:ABA in:Z + ZR in:
StemFlag leafStemFlag leaf
  1. Data used for calculations are from Figs 2–7. **Indicate correlation significance at P = 0.01 level (n = 24).

NSC remobilization0.94** 0.90**−0.84**−0.89**
14C in grains0.91** 0.83**−0.81**−0.83**
Photosynthetic rate−0.92**−0.84**0.89** 0.84**
Chlorophyll content−0.83**−0.85**0.69** 0.97**

The correlations of ABA and Z + ZR concentrations in the grains with grain filling rate during the linear growth period (9–39 DPA for WW and 9–33 DPA for WS plants, respectively) were also analysed (Fig. 8). The ABA concentration was positively correlated with grain filling rate with r = 0.97** (P = 0.01), whereas the correlation between the Z + ZR concentration and grain filling rate was insignificant (r = −0.32, P > 0.05).

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Figure 8. Relationships of grain filling rates with the concentrations of abscisic acid (ABA) (a) and zeatin (Z) + zeatin riboside (ZR) (b) in the grains of wheat. Data are from Figs 5, 6, and 7. Correlation coefficients (r) are calculated and asterisks (**) represent statistical significance at P = 0.01 (n = 32).

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Effects of exogenous ABA and kinetin

When ABA was applied to HN and WW pot-grown plants at early grain filling stage (9–13 DPA), Chl in the leaves was significantly reduced but the grain filling rate, remobilized carbon reserves in the stems, and grain weight were significantly increased (Table 4). The application of kinetin had the opposite effect.

Table 4.  Effects of exogenous ABA and kinetin on chlorophyll content of the flag leaves (mg g−1 FW), grain filling rate (mg grain−1 d−1), remobilization of carbon reserves (%) and grain weight (mg grain−1) of wheat
TreatmentChlorophyll content (n = 8)Grain filling rate (n = 8)Remobilized carbon reservea (n = 16)Grain weight (n = 16)
  1. Plants were grown in well-watered pots and treated with a high amount of nitrogen at heading. The leaves were sprayed either with 20 ×10−6 m ABA or with 40 ×10−6 m kinetin daily for 5 d starting at 9 d post-anthesis. Chlorophyll content was the mean of measurements made on 15, 21 and 27 d post-anthesis, respectively. The grain-filling rate was calculated according to Richards's (1959) equation. The remobilization of carbon reserves and grain weight were determined at maturity. a[Non-structural carbohydrate (NSC) in stems at anthesis − NSC  in  stems  at  maturity]/NSC  in  stems  at  anthesis × 100.  Statistical  comparison  was  within  the  same  column  and  the  same  cultivar. *, **Indicate significantly different with the control at P = 0.05 and P = 0.01 levels, respectively, according to an unpaired t-test.

Control4.770.632437.2
20 × 10−6 M ABA2.78**0.98*44**42.1*
40 × 10−6 M kinetin5.42*0.5512*34.9

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Our results showed that the remobilization of pre-stored assimilates was closely associated with senescence (Figs 2, 3 and 4). Water stress enhanced the senescence and led to more and faster remobilization of pre-stored carbon assimilates from the stem to grains. In contrast, HN slowed senescence and retarded such remobilization. It is believed that the whole-plant senescence in monocarpic plants initiates the remobilization of assimilates from vegetative tissues to grains (Nooden et al. 1997; Ori et al. 1999). However, Sinclair & Dewit (1975) stated that nutrient depletion from vegetative tissues to seeds or fruits sets plant senescence. The present study indicates that senescence and remobilization are coupled processes in wheat.

Although major plant hormones have been implicated in the senescence process, only cytokinins and ethylene have been shown definitely to have a role in regulation of senescence (Smart 1994). Our results showed that water stress substantially increased ABA accumulation in the leaves, stems, and grains, and markedly reduced Z + ZR in the leaves (Figs 6 & 7). ABA was significantly and negatively correlated with Pr and Chl of the flag leaves, whereas Z + ZR was positively correlated (Table 3). Exogenous ABA significantly reduced, while kinetin increased, the Chl content (Table 4). These results suggest that both ABA and cytokinins are involved in controlling senescence in wheat subjected to water stress. The mechanism in which ABA elicits senescence is probably that it increases ethylene synthesis or sensitivity, which induces plant senescence. It is noteworthy that ABA sharply increased as soon as the water deficit was implied, whereas Z and ZR showed almost no response to water-deficit treatment during the initial days (Figs 6 & 7). This implies that ABA may act as a more sensitive regulator than cytokinins in wheat plants under mild water stress.

We observed in present study that elevated ABA levels in WS stems were associated with the increase of 14C in the grains under WS treatments (Figs 3 & 7). ABA in both the leaves and stems, but not cytokinins (Z + ZR), was significantly and positively correlated with remobilization of pre-stored carbon (Table 3), and such remobilization was enhanced by exogenous ABA (Table 4). We conclude that enhanced remobilization by water stress during grain filling of wheat can be attributed, at least partly, to an elevated ABA level in the plant, although exogenously applied ABA could bring multiple effects such as stomatal closure which leads to reduction of photosynthesis.

Usually, water stress imposed during grain filling, especially at the early filling stage, can result in the reduction in grain weight (Nicolas et al. 1985a; Kobata, Palta & Turner 1992; Palta et al. 1994; Zhang et al. 1998; Wardlaw & Willenbrink 2000). The reduction is mainly attributed to the lowered number of endosperm cells, thereby decreased sink size per kernel (Singh & Jenner 1982; Nicolas, Gleadow & Dalling 1985b; Michihiro, Lui & Garvalho 1994). Our results showed that cytokinin contents in wheat grains showed a transient increase at early grain filling stage and reached a maximum at 6–9 DPA (Fig. 6), which coincided with the period of maximum endosperm cell division (Gleadow, Dalling & Halloran 1982; Gao et al. 1992). We observed that if a water stress was initiated at 9 DPA and controlled to a degree that plant could rehydrate overnight (Fig. 1), the maximum levels of Z + ZR in the grains were not affected (Fig. 6). The grain weight and grain yield were not significantly reduced by the controlled water deficit (Fig. 5, Table 1). We speculate that the division of endosperm cells was not affected under water stress in this experiment.

It is notable that the ABA concentration in the grains of WW plants was low at early grain filling sate (Fig. 7), and the grain-filling rate was slow and low grain weight happened when HN was applied (Fig. 5). Water stress markedly enhanced ABA accumulation in the grains, shortened the grain filling period, and increased the grain weight for the plant with HN. Enhanced ABA levels in the grains were consistent with the increase in grain filling rate under water stress. The ABA concentration in the grains, but not Z + ZR, was positively correlated with grain filling rate (Fig. 8). Very similar to the water stress, exogenous ABA significantly increased the grain filling rate and grain weight (Table 4). These results demonstrate that ABA accumulation in wheat grains can promote grain filling during grain development.

When wheat plants are subjected to water stress and N deficiency, their kernel numbers per spike will be reduced (e.g. Demotes-Mainard, Jeuffroy & Robin 1999). This did not happen in our study (Table 1). We believe mainly this is because our ‘stress’ treatments were mild and applied after the anthesis when the flower numbers per spike are already determined.

In conclusion, if a water deficit during the grain filling of wheat is controlled properly, plant can rehydrate overnight. A benefit from such a water deficit is that it can enhance plant senescence and lead to a fast and better remobilization of carbon from vegetative tissues to grains. The early senescence induced by the water deficit does not necessary reduce the grain weight even when the plants are grown under a NN condition. Furthermore, in situations in which plant senescence is unfavourably delayed such as by heavy use of nitrogen, the gain from the enhanced remobilization and accelerated grain filling rate may outweigh the loss of photosynthesis and shortened grain filling period and increase grain weight. Both ABA and cytokinins are involved in controlling the senescence. The enhanced remobilization and increased grain-filling rate are mainly attributed to elevated ABA levels in the stems and grains when subjected to water stress.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

This research was funded 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).

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  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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Received 8 May 2003; received in revised form 15 May 2003; accepted for publication 20 May 2003