The Effect of Plastic-Covered Ridge and

Journal of Integrative Agriculture© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.The Effect of Plastic-Covered Ridge and Furrow Planting on the Grain Filling and Hormonal Changes of Winter WheatLIU Yang *, HAN Juan *, WEN Xiao-xia, WU Wei, GUO Qiang, ZENG Ai and LIAO Yun-chengCollege of Agronomy, Northwest A&F University, Yangling 712100, P.R.ChinaAbstractAlthough plastic-covered ridge and furrow planting (RF) has been reported to produce substantial increases in the grain weight of winter wheat, the underlying mechanism is not yet understood. The present study used two cultivars, Xinong 538 and Zhoumai 18, and RF and traditional flatten planting (TF, control) with the objective of investigating the effect of RF on wheat grain filling and the possible relationship of hormonal changes in the wheat grains under RF to grain filling. The results indicated that RF significantly increased the grain weight, although the effects on grain filling were different: RF significantly increased the grain-filling rate and grain weight of inferior grains, whereas RF had no significant effect on grain-filling rate and grain weight of superior grains. The final grain weight of inferior grains under RF was 39.1 and 50.7 mg for Xinong 538 and Zhoumai 18, respectively, 3.6 and 3.4 mg higher than the values under TF. However, the final grain weight of superior grains under RF was only 0.6 and 0.8 mg higher than under TF for Xinong 538 and Zhoumai 18, respectively. RF significantly decreased the ethylene and gibberellic acid content in the inferior grains and increased the indole-3-acetic acid, abscisic acid and zeatin + zeatin riboside content in the inferior grains; however, no significant difference between RF and TF was observed for the hormonal content in the superior grains. Based on these results, we concluded that RF significantly modulated hormonal changes in the inferior grains and, thus, affected the grain filling and grain weight of the inferior grains; in contrast, RF had no significant effect on grain filling, grain weight and hormonal changes in the superior wheat grains.Key words: plastic-covered ridge and furrow planting, wheat, grain filling, hormoneINTRODUCTIONWinter wheat (Triticum aestivum L.) is the most im-portant food crop in the dryland region of northern China. However, the rainy season in this region does not coincide with the growth stages of winter wheat. In this region, more than 70% of the precipitation falls during the monsoon months from June to September (Xin and Wang 1998; Li et al . 2000), and, as a result, droughts are common during the growth stages ofwinter wheat. Hence, the key to increasing winter wheat productivity in this region lies in maximizing the utilization of precipitation and achieving the great-est possible increase in the water-use efficiency (WUE) of winter wheat (Li et al . 2001; Wang et al . 2001; Shao et al . 2007).Rainwater harvesting has been used for many years in semiarid areas to address the problems of water shortage (Zhu et al . 1994). Plastic-covered ridge and furrow planting (RF), a rainwater-harvesting system developed in the dryland region of northern China, hasReceived 8 September, 2012 Accepted 31 January, 2013Correspondence LIAO Yun-cheng, Tel: +86-29-87082990, E-mail: yunchengliao@ *These authors contributed equally to this study.1772LIU Yang et al.been considered to represent one of the most effective measures for increasing WUE and optimizing yields (Ren et al. 2008, 2010a). The results of many studies have indicated that, by collecting water from light rain and retaining surface runoff from heavy rain, this system could significantly improve soil moisture storage, prolong the period of moisture availability and enhance the production of agricultural crops (Li et al. 2001; Li and Gong 2002; Tian et al. 2003; Xie et al. 2005; Jia et al. 2006; Zhang et al. 2007). Accordingly, RF is widely employed in the dryland region of northern China.It has been reported that, in comparison to traditional flatten planting (TF), RF substantially increases the grain yield of such crops as winter wheat, maize (Zea mays L.) and millet (Panicum miliaceum L.) (Ren et al. 2008, 2010b; Liu et al. 2010). Furthermore, previous studies have suggested that RF can markedly increase the soil water content, soil nitrogen content and soil temperature, thereby increasing root development, the photosynthetic rate, the absorption and utilization of nutrients, and dry matter accumulation, thus improving grain yield (Li et al. 2001; Li and Gong 2002; Tian et al. 2003; Xie et al. 2005; Jia et al. 2006; Zhang et al. 2007; Ren et al. 2008; Liu et al. 2010).The yield potential of wheat is divided into three major components: grain weight, grain number per panicle and panicle number per plant. Grain filling, the final stage of growth in cereals in which the fertilized ovaries develop into caryopses, determines the grain weight (Yang and Zhang 2006). Modern high-yield crop production systems require high-yield outputs because of the increasing importance of improving grain filling (Saini and Westgate 2000; Zahedi and Jenner 2003). Although a previous study has indicated that RF significantly promotes the grain weight of winter wheat compared to TF, thus increasing the grain yield of winter wheat (Liu et al. 2010), no information is available on the effects of RF on the grain-filling process in winter wheat or the underlying mechanism. The grain filling of cereals is regulated by various factors, and plant hormones play an important role in regulating this process. Morrisetal et al. (1993) reported that, following pollination, zeatin (Z) and zeatin riboside (ZR) showed large transient increases in developing wheat grains. These increases coincided with the period of seed setting and maximum cell division in the endosperm. Higher abscisic acid (ABA) and lower ethylene (ETH) concentrations in wheat grains were associated with a higher filling rate, and the increase in the ratio of ABA to ETH was found to promote grain filling (Yang et al. 2006). The content of indole-3-acetic acid (IAA) was higher in superior grains than in inferior grains at the early grain-filling stage of rice (Oryza sativa L.) (Xu et al. 2007). The gibberellic acid (GA) content in wheat grain was also correlated with the grain-filling rate at the early grain-filling stage (Gao et al. 2000), whereas the maximal grain-filling rate was negatively correlated with GAs in rice grains (Yang et al. 2001). In addition, brassinosteroids (Wu et al. 2008) and polyamines (Yang et al. 2008) also affect grain filling in cereals.These studies indicate that hormones clearly affect wheat grain filling. However, the relationship between such hormonal changes and grain filling induced by RF remains unclear. The present study employed RF and TF cultivation systems to investigate the changes in IAA, ABA, Z+ZR, Gas, and ETH in wheat grains during the grain-filling process. The objective of the study was to investigate the effect of RF on the grain-filling process of winter wheat and how changes in endogenous hormones in the developing grains of winter wheat under RF cultivation are related to the grain-filling process.RESULTSYield and yield componentsThe RF system significantly increased the grain yield of the two cultivars (Table 1). Substantial increases of 14.6 and 11.9% for Xinong 538 and Zhoumai 18, respectively, were achieved compared with the TF treatment. However, RF planting had different effects on the yield components, significantly increasing the spikelets per panicle andThe Effect of Plastic-Covered Ridge and Furrow Planting on the Grain Filling and Hormonal Changes of Winter Wheat 1773grain weight of the two cultivars compared with TF. In contrast, there was no significant difference between RF and TF treatments for the panicles ha -1 of the two cultivars.Grain fillingRF had different effects on grain filling of superior and inferior grains, with RF planting significantly promoting the grain filling of inferior grains of thetwo cultivars (Fig. 1, Table 2). Both the maximum grain weight and the maximum and mean grain-filling rates of inferior grains of two cultivars were significantly higher than were those of the TF treatment. Conversely, RF had no significant effect on grain filling of superior grains of two cultivars. No significant difference was observed between the RF and TF treatments for the maximum grain weight or for the maximum and mean grain-filling rates of superior grains of the two cultivars.Table 1 Effect of RF on the yield and yield components of the winter wheatCultivar Treatment 1)Panicles ha -1 (×104)Spikelets per panicleGrain weight (mg)Grain yield (t ha -1)Xinong 538RF 454.6 a 36.9 a 41.8 a 7.0 a TF 451.3 a 33.7 b 40.3 b 6.1 b Zhoumai 18RF 416.7 a 34.9 a 51.7 a 7.5 a TF 414.5 a 32.8 b 49.1 b 6.7 b1)RF, plastic-covered ridge and furrow planting; TF, traditional flatten planting. The same as below.Values within a column and for the same cultivar followed by different letters are significantly different at P =0.05. The same as below.Fig. 1 Grain weights (A, Zhoumai 18; B, Xinong 538) and grain filling rates (C, Zhoumai 18; D, Xinong 538) of winter wheat under two cultivation methods. RF, plastic-covered ridge and furrow planting; TF, traditional flatten planting. s, superior grain; i, inferior grain.Vertical bars represent mean±SE (n=3). The same as below.1774 LIU Yang et al .Hormonal changesIAA IAA content in grains transiently increased at the early grain-filling stage, reaching a maximum at 12 d after anthesis for superior grains and 15 d after anthesis for inferior grains and decreasing thereafter (Fig. 2). IAA content in superior grains was significantly higher than that in inferior grains from 0 to 15 d post-anthesis. Although the two cultivars showed similar trends, IAA levels in superior and inferior grains of Zhoumai 18 were higher than those of Xinong 538 on the same day after anthesis.RF planting had different effects on IAA content of superior and inferior grains. During the 36 d after anthesis, no significant difference was observed between RF and TF treatments with regard to IAA content in superior grains. However, IAA content in inferior grains of RF treatment was significantly higher than that of TF treatment during the period of 0-15 d after anthesis, after which no significant difference was observed between RF and TF for the IAA content in the inferior grains.ABA Similar to IAA, ABA content in grains also transiently increased at the early grain-filling stage and then decreased (Fig. 3). However, ABA content ingrains reached a maximum at 15 d after anthesis for superior grains and 18 d after anthesis for inferior grains. ABA content in superior grains was significantly higher than that in inferior grains from 0 to 15 d after anthesis.RF planting had similar effects on ABA and IAA levels in superior and inferior grains. There was no significant difference between RF and TF treatments with regard to ABA content in superior grains during the grain-filling stage. However, ABA content in Zhoumai 18 inferior grains of RF treatment was significantly higher than that of TF during the grain-filling stage. In contrast, ABA content in Xinong 539 inferior grains of RF was significantly higher than that of TF treatment during 0-18 d after anthesis; thereafter, no significant difference was observed between RF and TF treatments for ABA content in inferior grains of Xinong 539.Z+ZR The change in pattern of Z+ZR content in winter wheat grains was similar to that of ABA and IAA. Z+ZR content in grains of two cultivars transiently increased at the early grain-filling stage,Table 2 Grain-filling characteristics of two winter wheat cultivars of Xinong 538 and Zhoumai 18 under two cultivation methodsCultivar Spikelet categoryTreatment A (mg)G max (mg grain -1 d -1)G mean (mg grain -1 d -1)D (d)Xinong 538SRF 42.1 a 4.21 a 1.25 a 18.3 a TF 41.5 a 4.12 a 1.26 a 18.6 a IRF 39.1 a 1.58 a 0.69 a 21.8 b TF 35.5 b 1.13 b 0.51 b 24.4 a Zhoumai 18S RF 53.7 a 4.46 a 1.58 a 20.9 a TF 52.9 a 4.38 a 1.51 a 21.8 a IRF 50.7 a 1.84 a 0.94 a 24.9 b TF47.3 b1.34 b0.71 b27.3 aA, the final grain weight (mg); G max , maximum grain-filling rates; G mean , mean grain-filling rates; D, the active grain-filling period. The same as below.Fig. 2 Effect of RF on the IAA content (A, Zhoumai 18; B,Xinong 538) in wheat grains.The Effect of Plastic-Covered Ridge and Furrow Planting on the Grain Filling and Hormonal Changes of Winter Wheat 1775reaching a maximum at 12 d after anthesis for superior grains and 15 d after anthesis for inferior grains and decreasing thereafter (Fig. 4). Z+ZR content in superior and inferior grains of Zhoumai 18 was higher than that of Xinong 538 on the same day after anthesis. In addition, Z+ZR content in superior grains was significantly higher than that in inferior grains prior to 12 d post-anthesis.RF treatment significantly affected Z+ZR content in grains. However, effects of RF on Z+ZR content in superior and inferior grains were different. Although Z+ZR content in superior grains in RF and TF treatm ents was not significantly different during the grain-filling stage, Z+ZR content in inferior grains of RF treatment was significantly higher than that of TF treatment during the 0-24 d following anthesis.GAs During the grain-filling stage, GA content in grains gradually decreased (Fig. 5). However, GA content in superior grains was significantly lower than that ininferior grains on the same day after anthesis. The two cultivars showed similar trends.Fig. 4 Effect of RF on Z+ZR content (A, Zhoumai 18; B, Xinong 538) in wheat grains.Fig. 5 Effect of RF on GAs content (A, Zhoumai 18; B, Xinong 538) in wheat grains.Fig. 3 Effect of RF on ABA content (A, Zhoumai 18; B, Xinong538) in wheat grains. ABTF-s RF-s TF-i RF-iTF-s RF-s TF-i RF-i1209060300120906030061218243036061218243036Days after antheis (d)G B s c o n t e n t (n g g -1 F W )1776 LIU Yang et al .RF treatment had a different effect on GA content in superior and inferior grains. During the grain-filling stage, there was no significant difference found between RF and TF treatments for GA content in superior grains. However, GA content in inferior grains of RF treatment was lower than that of TF during the grain-filling stage, and the difference between the two treatments was significant during the 0-15 d period after anthesis. The two cultivars showed similar trends.ETH Similar to GAs, ETH content in grains gradually decreased during the grain-filling stage, and ETH content in superior grains was significantly lower than thatFig. 6 Effect of RF on ETH content (A, Zhoumai 18; B, Xinong538) in wheat grains.ininferior grains on the same day after anthesis (Fig. 6). RF treatment also had a different effect on ETH content in superior and inferior grains. Although the RF treatment had no significant effect on ETH content in superior grains, RF treatment significantly decreased ETH content in inferior grains during the grain-filling stage. ETH content in inferior grains ofZhoumai 18 and Xinong 538 under RF treatment was significantly lower than that of TF prior to 21 and 15 d post-anthesis.Soil moisture and soil temperatureRF system significantly increased the soil moisture and soil temperature (Fig. 7). At anthesis, 20 d post-anthesis and maturity, the soil moisture and soil temperature of RF treatment were significantly higher than that of TF treatment.Fig. 7 Effects of RF on the soil moisture and soil temperature during the grain-filling stage. The soil temperature was recorded at 5, 10, 15, and 20 cm soil depths, and the average value was calculated. The soil moisture was measured from the 0 to 1.0 m profile layers. A, anthesis stage; F, 20 d post-anthesis; M, mature stage. As the results of the two cultivars were very similar, the data are presented as averages for the two cultivars. The vertical barsrepresent mean±SE (n=6).The Effect of Plastic-Covered Ridge and Furrow Planting on the Grain Filling and Hormonal Changes of Winter Wheat 1777positively and significantly correlated with the meangrain-filling rate (Table 3). These results indicatethat Z+ZR and IAA are involved in regulating wheatgrain filling. The changes in IAA and Z+ZR contentin grains showed very similar patterns. Z+ZR andIAA content in grains transiently increased at the earlygrain-filling stage and then decreased, and these twohormones reached a maximum at 12 d after anthesisfor superior grains and 15 d after anthesis for inferiorgrains. We also observed that the maximal IAA andZ+ZR content appeared immediately before the timeof the maximal grain-filling rate in both superior andinferior grains. Xu et al. (2007) suggested that CTKsregulate cell division in the endosperm of developingrice grains, and Davies (1987) stated that auxin alsostimulates cell division. High IAA levels in a sinkorgan could create an “attractive power”, leading toincreased cytokinin levels in grains (Seth and Waering1967; Singh and Gerung 1982). These results suggestthat IAA and Z+ZR may regulate wheat grain fillingat the early filling stage, most likely by manipulatingthe division of endosperm cells and, therefore, creatingsink strength.Table 3 Correlation coefficients of hormone contents in wheat grain with the final grain weights and the maximum and mean grain-filling rates of grainsCultivar1)Xinong 538Zhoumai 18G mean G max A G mean G max AIAA0.999**0.9190.938 1.000**0.9070.943 ABA0.997**0.986*0.965*0.996**0.985*0.976* Z+ZR0.984*0.972*0.986*0.996**0.983*0.980* GAs-0.921-0.917-0.906-0.910-0.904-0.911 ETH-0.993**-0.984*-0.974*-0.963*-0.974*-0.999** 1) IAA, indole-3-acetic acid; ABA, abscisic acid; Z, zeatin; ZR, zeatin riboside; GAs, gibberellins 1 plus 4; ETH, ethylene.* and **, significant at the 0.05 and 0.01 probability levels (n=4).In addition to Z+ZR and IAA, ABA and ETH also play important roles in regulating grain filling. Yang et al. (2006) suggested that the higher ABA concentration and lower ETH concentration found in wheat superior grains vs. inferior grains were associated with the higher filling rate in superior grains. Our present study found a similar result: ABA content in superio r grains was significantly higher than in inferior grains during the early grain stage (3-15 d after anthesis), and ETH content in superior grains was significantly lower than in inferior grains during the grain-filling stage. A regression analysis indicated that ABA content in grains was positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates; in contrast, ETH content in grains was negatively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. These results suggest that ABA promotes wheat grain filling and that ETH inhibits wheat grain filling, in agreement with a previous study (Yang et al. 2006).Several previous studies have reported that GAs are also involved in regulating grain development. Eeuwens and Schwabe (1975) suggested that the GADISCUSSIONRelationship of hormonal changes and the grain filling of wheatCytokinins (CTKs) play an important role in regulat-ing grain filling. It has been reported that the CTK levels in rice spikelets are significantly correlated with seed development (Yang et al. 2002; Zhang et al. 2009b). In barley (Hordeum vulgare L.), maize, rice, and wheat, high levels of cytokinins are generally found in endosperm of developing seeds and may be required for cell division during the early phase of seed setting (Michael and Seiler-Kelbitsch 1972; Saha et al. 1986; Morris et al. 1993; Dietrich et al. 1995; Yang et al. 2000). In addition to CTK, IAA also plays an important role in regulating grain filling (Yang and Zhang 2006; Xu et al. 2007; Zhang et al. 2009a). The present study indicated that Z+ZR content in grains was positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates; furthermore, the IAA conten t was1778LIU Yang et al.level was the highest in liquid endosperm of pea at the time of rapid pod elongation. A high GA level was also found in the large panicle of rice immediately before and at anthesis (Kurogochi et al. 1979; Suzuki et al. 1981). In the present study, GA content in superior grains was significantly lower than in inferior grains, although the GA content was not significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. These results suggest that, even though GAs may be involved in regulating wheat grain filling, these hormones may not be a determining factor for this process.Effect of RF on grain filling and grain weight in wheatIndeed, the amount of rainfall and its distribution have a profound impact on the crop production, environmental rehabilitation and economics of the dryland region of China (Ren et al. 2008). Frequent drought is also an important factor limiting crop yields, and RF is an effective way to increase water availability to increase crop yields (Ren et al. 2008). The present study indicated that RF planting significantly increased the grain yield of winter wheat (Table 1), a result that was similar to previous studies (Ren et al. 2008; Liu et al. 2010).The yield potential of wheat can be divided into three major components: grain weight, grain number per panicle and panicle number per plant. The present study indicated that RF significantly increases the grain weight and, thus, increases the grain yield. In addition, we found an interesting result: RF significantly increased the grain-filling rate and grain weight of inferior grains, whereas RF had no significant effect on superior grain filling and weight. Therefore, RF had different effects on the filling of superior and inferior grains of wheat. We found that, compared to TF, RF significantly decreases the ETH and GA contents in inferior grains and increases IAA, ABA and Z+ZR contents in inferior grains; however, RF had no significant effect on hormonal changes in superior grains. It is possible that these results explain why the RF treatment had different effects on grain filling of superior and inferior wheat grains. Xu et al. (2007) suggest that plastic mulching inhibits grain filling and decreases the grain weight of inferior rice grains but has no significant effect on filling and weight of superior rice grains. In addition, these authors found that the different effects of plastic mulching on filling of superior and inferior grains were related to the changes in ABA, IAA and ZR in grains, a result that was similar to the present study. Overall, RF primarily regulated the grain filling of inferior grains to alter the grain weight of wheat, and this regulation of grain filling in inferior grains was related to hormonal changes in grains.Many studies have suggested that RF can increase the moisture, temperature and nutrients in the soil, thus promoting crop growth and improving crop production (Li et al. 2001; Li and Gong 2002; Tian et al. 2003; Xie et al. 2005; Jia et al. 2006; Zhang et al. 2007; Ren et al. 2008; Liu et al. 2010). In the present study, we found that RF significantly increased the soil moisture and soil temperature during the grain-filling stage. Several previous studies have suggested that soil moisture and temperature were significantly correlated with the grain filling and grain weight of crops, indicating that effect of RF on wheat grain filling may be related to physicochemical characteristics of soil. The relationship between grain filling, hormonal changes, and soil physicochemical characteristics induced by RF should be studied further. CONCLUSIONCompared with TF, RF treatment significantly increased grain weight and spttikelets per plant of winter wheat, thereby increasing grain production. However, RF had different effects on grain filling and grain weight of superior and inferior grains of wheat: RF significantly increased grain-filling rate and grain weight of inferior grains but had no significant effect on grain filling and weight of superior grains. Compared with TF treatment, RF significantly decreased ETH and GAs contents in inferior grains and increased IAA, ABA and Z+ZR contents in inferior grains. However, no significant difference was observed for the hormonal content in superior grains between RF and TF. From these results, we conclude that RF system significantly regulatedThe Effect of Plastic-Covered Ridge and Furrow Planting on the Grain Filling and Hormonal Changes of Winter Wheat 1779hormonal changes in inferior wheat grains and thus affected the grain filling and grain weight of inferior wheat grains. In contrast, RF had no significant effect on grain filling, grain weight and hormonal changes of superior wheat grains.MATERIALS AND METHODSStudy site descriptionThi s study was conducted during 2011 and 2012 at the experimental station of Crop Specimen Farm in Northwest A&F University, Shaanxi Province, northwestern China. The station is located at a latitude of 34°20´N, a longitude of 108°24´E and an elevation of 466.7 m asl. The annual mean maximum and minimum air temperature at the site are 42 and -19.4°C, respectively, and the annual mean temperature is 12.9°C. The total annual sunshine duration is 2196 h, and the no-frost period is 220 d. The annual mean precipitation is 550 mm, with 70% falling from June to September. The soil in the top 1.2 m consists of Eum-Orthrosols (Chinese Soil Taxonomy), with a mean bulk density of 1.35 g cm-3. The readily available N, P and K are 58.43, 18.12 and 120.64 mg kg-1, respectively. The organic matter content of the topsoil (0-20 cm) and pH are 12.19 g kg-1 and 7.30, respectively.Experiment design and treatmentsTwo winter wheat cultivars, Zhoumai 18 and Xinong 538, were grown in the dryland field. The seeds were sown on October 10, 2011. Two treatments, RF and TF (control), were used for each cultivar. The RF system consisted of alternating ridges and furrows. The height of the ridges (or the depth of the furrows) was 15 cm. The RF system comprised six furrows and seven ridges. The length of the ridges and furrows was 6 m. The width of the plastic-covered ridges and the furrows was 0.20 and 0.30 m, respectively, and two rows of wheat were planted in the furrows with a row spacing of 0.25 m. Under the TF system, the wheat was planted in flat rows with a row spacing of 0.25 m. The sowing density was 150 kg ha-1. Each treatment had three replicates, with a completely randomized block design. The plot measured 6 m×3.25 m. For fertilization, 150 kg ha-1 urea and 150 kg ha-1 diammonium orthophosphate were applied at basal levels. No irrigation was conducted during the winter wheat growing season.Sampling and measurement400 spikes that flowered on the same day were chosen and tagged in each plot. Twenty tagged spikes from each plot were then sampled at 3-d intervals from anthesis to maturity. All the grains from each spike were removed, and the grains for each spike were divided into superior and inferior grains. The most basal grains in middle spikelets (4-12 spikelets) from the bottom of a spike were considered superior grains, and the most distal grains in middle spikelets (4-12 spikelets) from the bottom of a spike were considered inferior grains (Jiang et al. 2003). Half of the sampled grains were used for the hormone measurements; the other half were dried at 70°C to a constant weight, and the weight was recorded.Grain-filling processThe grain-filling process was fitted using Richards’s (1959) growth eq., as described by Zhu et al. (1988):NktBAW1)e1(−+= (1) The grain-filling rate (G) was calculated as the derivative of eq. (1):)1()1(NNktktBeAkBeG+−−+= (2)Where, W, the grain weight (mg); A, the final grain weight (mg); t, time after anthesis (d); B, k and N, coefficients determined by regression.The active grain-filling period was defined as the period when W was between 5% (t1) and 95% (t2) of A. The average grain-filling rate during this period was therefore calculated from t1 to t2.HormonesThe methods for the extraction and purification of Z+ZR, GAs (GA1+GA4), IAA, and ABA were essentially identical to those described by Yang et al. (2001). A sample of approximately 0.5 g (tiller buds approximately 0.2 g) was ground in a mortar (on ice) with 5 mL 80% (v/v) methanol extraction buffer containing 1 mmol L-1 butylated hydroxytoluene (BHT) as an antioxidant. The methanolic extracts were incubated at 4°C for 4 h and centrifuged at 10000×g for 15 min at 4°C. The supernatants were passed through Chromosep C18 columns (C18 Sep-Park Cartridge, Waters Corp, USA) that had been prewashed with 10 mL 100% and 5 mL 80% methanol. The hormone fractions were dried under N2 and dissolved in 1 mL phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5) for analysis with an enzyme-linked immunosorbent assay (ELISA).The mouse monoclonal antigens and antibodies against Z+ZR, GAs (GA1+GA4), IAA, ABA, and immunoglobulin G-horseradish peroxidase (IgG-HRP) used in the。