Effects of conservation tillage on grain filling and hormonal changes in wheat under simulated rainfall conditions

Field Crops Research 144 (2013) 43–51

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Effects of conservation tillage on grain filling and hormonal changes in wheat under simulated rainfall conditions Yang Liu a,1 , Yanwei Sui a,1 , Dandan Gu b , Xiaoxia Wen a , Yu Chen a , Changjiang Li a , Yuncheng Liao a,∗ a b

College of Agronomy, Northwest A&F University, Yangling 712100, China College of Plant Protection, Northwest A&F University, Yangling 712100, China

a r t i c l e

i n f o

Article history: Received 12 November 2012 Received in revised form 5 January 2013 Accepted 5 January 2013 Keywords: Conservation tillage Grain filling Hormone Wheat Rainfall

a b s t r a c t The objective of this study was to investigate the effect of conservation tillage (CT) on wheat grain filling under different precipitation levels and the relationship with endogenous hormonal changes. In the present study, CT was compared to traditional bare soil tillage (BT) under different precipitation conditions simulated during the wheat growth stage, and the resulting hormonal changes in the grains were measured. The results indicated that the effect of CT on wheat grain filling is significantly related to precipitation. At the 220-mm rainfall level, CT significantly promoted grain filling of the superior and inferior grains. CT also significantly promoted the grain filling of inferior grains at 260-mm rainfall yet had no significant effect on filling of the superior grains. In contrast, CT had no significant effect on the grainfilling rates and grain weight of superior and inferior grains at the 300-mm rainfall level. At 220- and 260-mm rainfall, CT significantly increased the indole-3-acetic acid (IAA) and zeatin (Z) + zeatin riboside (ZR) contents and decreased the abscisic acid (ABA), ethylene (ETH), and gibberellic acid (GA) contents in the inferior grains, whereas CT only had these effects in the superior grains at the 220-mm rainfall level. The IAA, ABA, and Z + ZR contents in the grains were positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates; the ETH concentration was negatively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. In addition, both precipitation and CT significantly increased the soil moisture during the grain-filling stage. Based on these results, we conclude that the CT treatment significantly increased the soil moisture, thus regulating the grain filling of wheat, and that this process was significantly related to the balance of hormones in the grains. © 2013 Elsevier B.V. All rights reserved.

1. Introduction China is one of the largest agricultural countries of the world, with approximately 140 million ha of agricultural lands, including a large dryland region in the north that accounts for approximately 56% of the nation’s total land area (Xin and Wang, 1998). Although winter wheat (Triticum aestivum L.) is one of the most important food crops in this region, the region’s rainy season does not coincide with the growth stage for winter wheat. Over 70% of the precipitation falls during the monsoon months from June to September (Li et al., 2000) and, as a result, droughts are a common occurrence during the winter wheat growth stage. Hence, the key to increasing winter wheat productivity in this region lies in maximizing the conservation and utilization of the soil water and achieving the largest possible increase in winter wheat water-use efficiency

∗ Corresponding author. Tel.: +86 029 87082990. E-mail addresses: [email protected], [email protected] (Y. Liao). 1 These authors contributed equally to this study. 0378-4290/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fcr.2013.01.009

(WUE) (Li et al., 2001). Many studies have indicated that conservation tillage (CT), including no tillage and straw mulching, conserve soil water, decrease soil water evaporation, and substantially promote the WUE of crops (Norton, 2008; Moraru and Rusu, 2010; Martinez et al., 2011; Zhang et al., 2011; Blanco-Moure et al., 2012). In addition, CT can provide some extra benefits, such as improving soil structure and raising soil organic matter levels (Peng and Horn, 2008; Rusu et al., 2009; Moraru and Rusu, 2010). Because of these advantages, CT is widely utilized in the dryland regions of northern China. It has been reported that CT can increase the grain yield of crops when compared with traditional bare soil tillage (BT) (Chen et al., 2011; Tang et al., 2011); however, there are reports that CT reduces, rather than increases, grain yields (Xie et al., 2008; Kirkegaard, 1995; Su et al., 2007). These previous studies have suggested that the effect of CT on grain yield may be related to the soil water content, soil temperature, soil nutrition, and other such factors (Wang et al., 2007). However, the biochemical mechanism underlying the increase or reduction in grain yield under CT has not yet been elucidated.

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The yield potential of wheat can be dissected 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 fertilized ovaries develop into caryopses, determines the grain weight (Yang and Zhang, 2006). However, in modern crop production systems, with their high-yield outputs, improvement in grain filling has become more challenging than ever (Saini and Westgate, 2000; Zahedi and Jenner, 2003). Thus, although it is important to determine whether and how CT affects wheat grain filling, no information is available regarding the effects of CT on the grain-filling process of winter wheat and the underlying biochemical mechanism. Grain filling in cereals is regulated by various factors, and plant hormones play an important role in modulating those factors and the grain-filling process. Morris et al. (1993) have reported that zeatin (Z) and zeatin riboside (ZR) in developing wheat grains present large transient increases following pollination that coincide 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 have been associated with a higher filling rate, and an increase in the ratio of ABA to ETH promotes grain filling (Yang et al., 2006). The content of indole-3-acetic acid (IAA) has been found to be higher in superior grains than in inferior grains in the early grain-filling stage of rice (Xu et al., 2007). The gibberellic acid (GA) content in wheat grains is also correlated with the grain-filling rate at the early grain-filling stage (Gao et al., 2000), and the maximal grain-filling rate was negatively correlated with GA in rice grains (Yang et al., 2001). In addition, brassinosteroids (Wu et al., 2008) and polyamines (Yang et al., 2008) also affect the grain filling of cereals. These studies indicate that the hormones markedly affect wheat grain filling; however, the relationship between the hormonal changes in wheat grains and the grain filling induced by CT remains unclear. Some previous studies have suggested that the effect of CT on grain yield is related to precipitation (Wang et al., 2001, 2007). Ren et al. (2008) suggested that rainfall collection with ridge and furrow cultivation (RC), a CT practice, significantly increases corn (Zea mays L.) yield at 230-mm and 340-mm precipitation levels; however, no significant difference was observed for the corn yield between RC and the conventional flat practice at a 440-mm precipitation level. Gao and Li (2005) have indicated that straw mulching significantly increases the grain yield of dryland wheat at lower precipitation levels, whereas straw mulching decreases the grain yield of dryland wheat at higher precipitation levels. Dong (2011) has found that straw mulching increases the grain yield of wheat under no-irrigation conditions, whereas straw mulching decreases the grain yield of wheat under irrigation conditions when the irrigation level is 40 mm. In the dryland region of northern China, the annual precipitation is significantly different among different sites, and the average coefficient of variation for the annual precipitation levels of different sites in this region of northern China is 40–50% (Xin and Wang, 1998). Accordingly, research into the effect of CT on grain yields under different precipitation conditions in the dryland region of northern China and in similar regions and the underlying mechanism is necessary for the rational utilization of CT systems in dryland regions. In the present study, different tillage practices and different precipitation levels during the growth stage of winter wheat were used, and the hormonal changes in the grains during filling were measured. The objective of the study was to investigate the relationship between the effect of CT on grain filling in wheat and precipitation during the winter wheat growth stage and to determine how the changes in endogenous hormones in the developing grains of wheat under CT are related to the grain-filling process. These data will provide some reference for the rational utilization of CT in dryland regions.

2. Materials and methods 2.1. Study site description This study was conducted from 2010 to 2012 at the experimental station of the Crop Specimen Farm in Northwest A&F University, Shaanxi Province, northwestern China: latitude of 34◦ 20 N, longitude of 108◦ 24 E and an elevation of 466.7 m above sea level. The annual mean maximum and minimum air temperatures at the site during the study period were 42 ◦ C and −19.4 ◦ C, respectively, and the annual mean temperature was 12.9 ◦ C. The total yearly sunshine duration was 2196 h, and the no-frost period was 220 days. The annual mean precipitation was 550 mm, with 70% of that precipitation falling between June and September. The top 1.2 m of soil was Eum-Orthrosols (Chinese soil Taxonomy), with a mean bulk density of 1.35 g cm−3 . The readily available N, P, and K concentrations were 58.43, 18.12, and 120.64 mg kg−1 , respectively. The organic matter content of the first 0–20 cm of topsoil and pH were 12.19 g kg−1 and 7.30, respectively. 2.2. Experimental design and treatments The experiment was performed in large-sized waterproof sheds. The internal shed dimensions were 32 m (length) × 15 m (width) × 3 m (height). The sheds had a transparent plastic-covered roof and four open sides. The mobile sheds were used to control natural rainfall on rainy days. The experiment was a 3 × 2 (3 levels of rainfall and 2 levels of tillage practice) factorial design, with 6 treatment combinations. Each of the treatments contained three plots as repetitions in a complete randomized block design. Three rainfalls levels, 220, 260, and 300 mm, were simulated during the winter wheat growth stage. For each rainfall level, two tillage practices were utilized: CT, no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight; and BT, plow tillage was conducted, and no straw was mulched. One winter wheat cultivar, Xinong 979, was grown in a dryland field. The seeds were sown on October 20 for the 2010 growth year and October 21 for 2011. The sowing density was 150 kg ha−1 , with a row spacing of 0.25 m. Fertilizer at 150 kg ha−1 urea and 150 kg ha−1 diammonium orthophosphate was applied at basal levels. No irrigation was conducted during the winter wheat growing season. In this experiment, we adopted the use of a rainfall simulator (RS) to supply the crop water requirements, and no natural rainfall occurred during the corn growing season. The RS was used according to Ren et al. (2008). In the rainfall simulation, three total seasonal rainfalls, 220, 260, and 300 mm, corresponded to light, moderate, and heavy natural rain levels. This rainfall level partitioning was derived from the spatial and temporal characteristics of the rainfall distribution in the semi-arid regions of northern China over the past 50 years. A rainfall event of 10–20 mm is considered a substantial rainfall in the semi-arid regions in China. Approximately 80% of the natural rainfall events in the 50-year period were in the range of 5–50 mm, and the I10 (maximum rainfall intensity in 10 min) was 30 mm h−1 , which is not a constant but an averaged value determined using a standard rain gauge over three consecutive weeks. The data analysis also indicated that approximately 80% of the total amount of annual rainfall resulted from rainstorms producing over 10 mm of rainfall (Wang and Zhai, 2003). Therefore, the minimum and maximum individual rainfall events in the experiment were 15 mm and 36 mm, respectively. Over 10 mm of rainfall was divided into sub-rainfall levels with equal to or less than 10 mm of rainfall and with approximately 2-h intervals between each rainfall event. A detailed description of the rainfall determination is shown in Table 1. In this simulation experiment, the partition of

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Table 1 Partition of rainfall simulation during winter wheat-growing seasons. Growth stage

Rainfall events

Rainfall duration

Seedling Wintering

1 2

Green Jointing Grain filling

1 1 3

Maturing

1

29–30 October 15–16 December 19–20 January 27–28 February 16–17 March 27–28 April 10–11 May 24–25 May 5–6 June

the application volume in pulsed rainfall events was not completely realistic under field conditions but was reasonably close. 2.3. Sampling and measurements Four hundred spikes that flowered on the same day were chosen and tagged in each plot. Twenty tagged spikes from each plot were sampled at 3-d intervals from anthesis to maturity, and all the grains from each spike were removed. The grains on a spike were divided into superior grains and inferior grains: the most basal grains in the middle spikelets (4–12 spikelets) from the bottom of a spike were considered superior grains, and the most distal grains in the 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 measurements of hormones, and the other half was dried at 70 ◦ C to a constant weight and weighed. 2.3.1. Grain-filling process The grain-filling process was fitted using Richards’s (1959) growth equation, as described by Zhu et al. (1988): W=

A (1 + Be−kt )

1/N

(1)

The grain-filling rate (G) was calculated as the derivative of Eq. (1): G=

AkBe−kt (1 + Be−kt )

(N+1)/N

(2)

W is the grain weight (mg); A is the final grain weight (mg); t is the time after anthesis (d); B, k and N are the 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 . 2.3.2. Hormones The 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 bud of 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 10,000 × g for 15 min at the same temperature. The supernatants were passed through Chromosep C18 columns (C18 Sep-Park Cartridge, Waters Corp, USA) and prewashed with 10 mL 100% and then 5 mL 80% methanol. The hormone fractions were dried with 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 enzyme-linked immunosorbent assay (ELISA) evaluation. The antigens and mouse monoclonal antibodies against Z + ZR, GAs (GA1 + GA4 ), IAA, ABA, and immunoglobulin G-horseradish

Daily rainfall distribution (mm) 220

260

300

25 15 16 16 27 28 28 30 35

35 18 22 19 32 33 33 33 35

50 22 28 22 36 36 36 35 35

peroxidase (IgG-HRP) used in the ELISA were produced at the Phytohormones Research Institute, China Agricultural University, China. The quantification method for Z + ZR, GAs (GA1 + GA4 ), IAA, and ABA by ELISA was described previously (Yang et al., 2001). The recovery rates of IAA, Z + ZR, ABA, and GAs were 86.3 ± 4.5%, 89.1 ± 4.4%, 87.2 ± 3.6%, and 79.8 ± 5.1%, respectively. The level of ethylene evolved from the analyzed grains was determined according to the method described by Beltrano et al. (1994), with modifications. Briefly, the sampled grains were placed between two sheets of moist paper for 1 h at 27 ◦ C in darkness to allow the wound-induced ethylene production to subside. Each sample contained 80–100 grains. The grains were then transferred to 25-ml glass vials containing moist filter paper, and the vials were immediately sealed with airtight Suba-Seal stoppers and incubated in the dark for 8 h at 27 ◦ C. A 1-ml gas sample was withdrawn through the Suba-Seal using a gas-tight syringe, and the ethylene level was assayed using a gas chromatograph (Trace GC UItraTM , Thermo Fisher Scientific, USA) equipped with a Porapak Q column (0.3 cm × 200 cm, 0.18–0.30 mm) and a flame ionization detector (FID). The temperatures for the injection port, column, and detector were kept constant at 70, 70, and 150 ◦ C, respectively. Nitrogen was used as a carrier gas at a flow rate of 40 kPa, and hydrogen and air were used for the FID measurement at rates of 35 and 350 ml min−1 , respectively. The rate of ethylene evolution was expressed as a function of the unit fresh weight (FW). 2.4. Yield and yield components Plants (except for border plants) from a 2-m2 site from each plot were harvested at maturity for the determination of the grain yield. The yield components, i.e., the spikes per square meter, grain number per spike and grain weight, were determined from plants harvested from a 1-m2 site (excluding the border plants) randomly sampled from each plot. 2.5. Soil moisture The soil moisture was measured on April 25, May 5, May 20, and June 10. The soil moisture was determined gravimetrically to a depth of 100 cm at 10-cm increments in the topsoil (0–40 cm) and at 20-cm increments below the topsoil (40–100 cm). Five measurement points were selected in each plot. The measurement of the soil moisture used oven-drying and was performed according to the method described by Bao (2007). 2.6. Statistical analysis SPSS 16.0 was used for an ANOVA. The data from each sampling were analyzed separately. The means were tested using the least significant difference at P0.05 (LSD0.05 ). The hormone levels are presented as the data from 2011 to 2012.

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Table 2 Effects of conservation tillage and precipitation on grain yield and yield component of winter wheat. Year

Rainfall (mm)

Tillage

No. of panicles (×104 hm−2 )

Spikelets per panicle

Grain weight (mg)

Grain yield (t hm−2 )

2010–2011

220

BT CT BT CT BT CT

355.1e 370.3d 399.4c 409.5b 415.7ab 417.1a

25.8e 30.6d 33.8c 37.1b 38.7ab 41.1a

33.7d 40.2c 42.3b 45.1a 45.1a 44.7a

3.1e 4.6d 5.7c 6.9b 7.2ab 7.7a

BT CT BT CT BT CT

350.9e 366.5d 393.2c 401.7b 411.8a 416.0a

27.1e 32.2d 34.3c 37.7b 39.4ab 42.3a

35.1d 40.7c 42.6bc 44.9a 45.3a 44.1ab

3.3e 4.8d 5.7c 6.8b 7.3a 7.8a

260 300 2011–2012

220 260 300

Values within a column and for the same year followed by different letters are significantly different at P = 0.05. CT: conservation tillage, no tillage was conducted and corn straw was mulched at 7000 kg hm−2 in dry weight. BT: traditional bare soil tillage, plow tillage was conducted and no straw was mulched.

3. Results 3.1. Yield and yield component Precipitation significantly increased the grain yield of the wheat, with the wheat grain yield significantly increasing with an increase in the precipitation (Table 2). The CT system also affected the grain yield, although the effect of CT on the grain yield was markedly related to the amount of precipitation during the wheat growth stage. The CT practice significantly increased the grain yield at the 220- and 260-mm rainfall levels, whereas there was no significant difference observed between BT and CT for the grain yield at 300mm rainfall. Similarly, CT and the precipitation level also significantly affected the yield components: with increasing precipitation, the panicles per ha, spikelets per panicle and grain weight all were

significantly increased. At the 220- and 260-mm rainfall levels, the panicles per ha, spikelets per panicle, and grain weight of the CT treatment were significantly higher than that of the BT treatment; in contrast, there was no significant difference observed between BT and CT for the panicles per ha, spikelets per panicle, and grain weight at the 300-mm rainfall level. 3.2. Grain filling CT had a different effect on grain filling at the different precipitation levels. At the 220-mm rainfall level, CT significantly increased the maximum grain weights and the maximum and mean grainfilling rates of the superior grains and inferior grains compared to the BT treatment (Fig. 1 and Table 3). At 260-mm rainfall, CT significantly increased the maximum grain weights and the maximum and mean grain-filling rates of the inferior grains, whereas CT had

Fig. 1. The effects of conservation tillage on grain weights (A, 220-mm rainfall level; B, 260-mm rainfall level; C, 300-mm rainfall level) and grain-filling rates (D, 220-mm rainfall level; E, 260-mm rainfall level; F, 300-mm rainfall level) of winter wheat at different rainfall levels. S, superior grains; I, inferior grains. BT: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The vertical bars represent the ± the standard error of the mean (n = 3).

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Table 3 Grain-filling characteristics of winter wheat under different tillage and precipitation.

Superior grain

Rainfall (mm)

Tillage

Wmax (mg)

Gmean (mg per grain d−1 )

Gmax (mg per grain d−1 )

220

BT CT BT CT BT CT

39.1d 41.6c 46.1b 46.7ab 47.5a 47.3a

0.89c 1.22b 1.37a 1.43a 1.43a 1.43a

1.77c 2.15b 2.36a 2.41a 2.49a 2.49a

BT CT BT CT BT CT

34.2d 38.0c 41.4b 44.3a 44.6a 43.9a

0.15e 0.39d 0.51c 0.70b 0.82a 0.84a

0.29d 0.63c 0.92b 1.28a 1.31a 1.36a

260 300

Inferior grain

220 260 300

Values within a column and for the same grain type followed by different letters are significantly different at P = 0.05. CT: conservation tillage, no tillage was conducted and corn straw was mulched at 7000 kg hm−2 in dry weight. BT: traditional bare soil tillage, plow tillage was conducted and no straw was mulched. Wmax : the final grain weight; Gmax : maximum grain-filling rates; Gmean : mean grain-filling rates.

Fig. 2. The effects of conservation tillage on the IAA content in wheat grains under different rainfall levels (A, 220-mm rainfall level; B, 260-mm rainfall level; C, 300-mm rainfall level). S, superior grain; I, inferior grain. BT: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The vertical bars represent the ± the standard error of the mean (n = 3).

no significant effects on the superior grains. Conversely, there were no significant differences observed between CT and BT for the maximum grain weights and the maximum and mean grain-filling rates of the superior grains and inferior grains at the 300-mm rainfall level. 3.3. Hormonal changes 3.3.1. IAA and Z + ZR The IAA and Z + ZR contents in the grains presented the same pattern of change during grain filling. The IAA and Z + ZR contents in the grains transiently increased during the early grain-filling stage and reached a maximum at 12 days after anthesis for the

superior grains and 15 days after anthesis for the inferior grains before decreasing thereafter (Figs. 2 and 3). The IAA and Z + ZR contents in the superior grains were significantly higher than in the inferior grains from 0 to 12 days post-anthesis. CT had a different effect on the IAA and Z + ZR contents in the superior grains and inferior grains at the different precipitation levels. CT significantly increased the IAA and Z + ZR contents in the superior and inferior grains at the 220-mm rainfall level, and the IAA and Z + ZR contents in the superior and inferior grains were significantly higher than in the BT treatment at 3–15 days after anthesis. CT also significantly increased the IAA and Z + ZR contents in the inferior grains at 260-mm rainfall, whereas CT had no such effect in the superior grains. In contrast, there was no significant

Fig. 3. The effects of conservation tillage on the Z + ZR content in wheat grains under different rainfall levels (A, 220-mm rainfall level; B, 260-mm rainfall level; C, 300-mm rainfall level). S, superior grain; I, inferior grain. BT: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The vertical bars represent the ± the standard error of the mean (n = 3).

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Fig. 4. The effects of conservation tillage on the ABA content in wheat grains under different rainfall levels (A, 220-mm rainfall level; B, 260-mm rainfall level; C, 300-mm rainfall level). S, superior grain; I, inferior grain. BT: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The vertical bars represent the ± the standard error of the mean (n = 3).

Fig. 5. The effects of conservation tillage on the ETH evolution rate in wheat grains under different rainfall levels (A, 220-mm rainfall level; B, 260-mm rainfall level; C, 300-mm rainfall level). S, superior grain; I, inferior grain. BT: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The vertical bars represent the ± the standard error of the mean (n = 3).

difference observed between CT and BT for the IAA and Z + ZR contents in the superior and inferior grains at the 300-mm rainfall level. 3.3.2. ABA Similar to IAA and Z + ZR, the ABA content in the grains also transiently increased in the early grain-filling stage and then decreased (Fig. 4). However, the ABA content in the grains reached a maximum at 15 days post-anthesis for the superior grains and 18 days post-anthesis for the inferior grains. The ABA content in the superior gains was significantly higher than that in the inferior grains from 0 to 15 days post-anthesis. CT significantly affected the ABA content in the grains, though the effect of CT on the ABA content in grains was opposite to what was observed for IAA and Z + ZR. At the 220-mm rainfall level, CT significantly decreased the ABA content in the superior and inferior grains at 3–18 days post-anthesis; CT also significantly decreased the ABA content in the inferior grains at 260-mm rainfall but had no such effect in the superior grains. In contrast, there was no significant difference observed between CT and BT with regard to the ABA content in the superior and inferior grains at the 300-mm rainfall level. 3.3.3. ETH and GAs The GA and ETH contents in the grains displayed a similar pattern of change during grain filling, with the GA and ETH contents in the grains decreasing gradually (Figs. 5 and 6). The GA content in the superior grains was significantly lower than that in the inferior grains at 3–12 days post-anthesis. However, the ETH content in the superior grains was significantly lower than that in the inferior grains during the entire grain-filling stage.

Similar to the ABA content, at the 220-mm rainfall level, CT significantly decreased the ETH and GA contents in the superior and inferior grains. CT also significantly decreased the GA and ETH contents in the inferior grains at 260-mm rainfall, whereas no such effect was found for the superior grains. There was no significant difference observed between CT and BT for the GA and ETH contents in the superior and inferior grains at the 300-mm rainfall level. 3.4. Soil moisture The amount of precipitation significantly increased the soil moisture during the grain-filling stage: the soil moisture displayed a significant increasing trend with increasing precipitation (Fig. 7). In addition, CT also significantly affected the soil moisture. Although CT significantly increased the soil moisture compared to BT at each rainfall level, the difference in the soil moisture between CT and BT declined with increasing rainfall. 4. Discussion 4.1. Effects of conservation tillage and precipitation on grain yield Agricultural production in the arid farming regions of China is highly dependent on rainfall, and the seasonal rainfall amount and its distribution have a profound impact on the crop production, environmental rehabilitation, and economics of the region (Ren et al., 2008). Frequent drought is also an important factor that limits crop yields. Previous studies have suggested that CT could improve the moisture, temperature, and the nutrients of soil and that the CT system is an effective way to increase water availability for crop yield (Blanco-Moure et al., 2012; Martinez et al., 2011; Moraru and

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Fig. 6. The effects of conservation tillage on the GA1+4 content in wheat grains under different rainfall levels (A, 220-mm rainfall level; B, 260-mm rainfall level; C, 300-mm rainfall level). S, superior grain; I, inferior grain. BT: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The vertical bars represent the ± the standard error of the mean (n = 3).

Fig. 7. The effects of conservation tillage on soil moisture during the grain-filling stage at different rainfall levels. A1, A2, and A3 correspond to the 220-, 260-, and 300-mm rainfall levels, respectively. B1: traditional bare soil tillage – plow tillage was conducted, and no straw was mulched. CT: conservation tillage – no tillage was conducted, and corn straw was mulched at 7000 kg hm−2 dry weight. The soil moisture was measured on April 25, May 5, May 20, and June 10 during the grain-filling stage. The data are presented as the averages for the measured values on April 25, May 5, May 20, and June 10. The vertical bars represent the LSD0.05 .

Rusu, 2010; Norton, 2008; Zhang et al., 2011). However, the effect of CT on grain yield is disputed. Some studies have indicated that CT significantly increases grain yield (Chen et al., 2011; Tang et al., 2011), whereas other studies have found that CT reduces, rather than increases, grain yield (Xie et al., 2008; Kirkegaard, 1995; Su et al., 2007). In the present study, CT significantly promoted the grain yield of winter wheat at the 220- and 260-mm rainfall levels; however, the CT system had no significant effect on the wheat grain yield at 300-mm rainfall. This result indicates that the effect of CT on the grain yield of winter wheat is related to precipitation during the winter wheat growth stage and is similar to the results of previous studies on maize and plastic-covered ridge and furrow planting, another conservation tillage practice. Therefore,

Table 4 Correlation coefficients of peak hormone contents in wheat grain with the maximum grain filling rate (Gmax ), mean grain filling rate (Gmean ), and maximum grain weight (Wmax ) of winter wheat.

IAA ABA Z + ZR ETH GAs IAA/ETH Z + ZR/ETH ABA/ETH

Wmax

Gmean

Gmax

0.788** 0.613* 0.927** −0.863** 0.205 0.773** 0.835** 0.682*

0.988** 0.591* 0.926** −0.995** −0.292 0.962** 0.942** 0.940***

0.983** 0.607* 0.906** −0.986** −0.351 0.952** 0.927** 0.944**

IAA, indole-3-acetic acid; ABA, abscisic acid; Z, zeatin; ZR, zeatin riboside; GAs, gibberellins 1 plus 4; ETH: ethylene; Wmax : the final grain weight (mg); Gmax : maximum grain-filling rates; Gmean : mean grain-filling rates. * Significant at the 0.05 probability level (n = 12). ** Significant at the 0.01 probability level (n = 12).

the utilization of CT in a dryland region should take into consideration the precipitation of the local region. Based on the results of the present study, we suggest that, in the case of wheat, the adoption of CT practices, including no tillage and straw mulching, in areas receiving 220–300 mm rainfall will provide benefits during the entire growth period. The yield potential of wheat can be dissected into three major components: grain weight, grain number per panicle, and panicle number per plant. Grain filling, which determines the grain weight, is an important agronomic trait of wheat, and previous studies have indicated that CT significantly affects the grain weight of wheat (Liu et al., 2004; Zhang et al., 2009b). Wheat grains can be divided into superior and inferior grains according to the degree and rate of filling. Yang and Zhang (2010) suggested that “super” rice cultivars frequently do not exhibit their high yield potential due to the poor grain filling of inferior grains. In the present study, we found that the effect of CT on grain filling in wheat is significantly related to the precipitation level, with CT having a different effect on the filling of superior and inferior grains at different precipitation levels. At the 220-mm rainfall level, CT significantly increased the grain-filling rates of the superior and inferior grains and, thereby, increased the wheat grain weight. CT also significantly increased the wheat grain weight at the 260-mm rainfall level. However, CT had no significant effect on the grain-filling rate and grain weight of the superior grains, only significantly increasing the grain-filling rate and grain weight of the inferior grains. Conversely, CT had no significant effect on the grain-filling rates and grain weights of the superior and inferior grains at the 300-mm rainfall level. These results indicate that the effect of CT on grain weight had a more significant relationship with the grain filling of inferior grains. The differing effects of CT on the wheat grain weight at different precipitation levels was mainly

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due to the different effects on the filling of the inferior grains at the different precipitation levels. A previous study suggests that soil moisture may have different effects on the filling of superior and inferior grains (Xu et al., 2007). Xu et al. (2007) have suggested that non-flooded plastic mulching cultivation significantly decreases the grain-filling rate and grain weight of inferior grains of rice compared to traditional flooding cultivation; however, this cultivation has no such effect on superior rice grains, indicating that the inferior grains are more sensitive to soil moisture. The results of the present study indicate that the precipitation level and CT both significantly affect soil moisture. Thus, we suggest that the reason CT had a different effect on the filling of superior and inferior grains at different precipitation levels may be that the soil moisture was sufficient for wheat grain filling at 300-mm rainfall; accordingly, CT had no significant effect on the filling of the superior and inferior grains, even though it significantly increased the soil moisture. In contrast, as the soil moisture was significantly decreased at the 260-mm rainfall level, the inferior grain filling was inhibited because these grains were more sensitive to soil moisture. CT significantly increased the soil moisture, thus promoting inferior grain filling, whereas the filling of the superior grains presented no significant change. At the 220-mm rainfall level, the soil moisture was seriously insufficient for grain filling, such that the filling of both the superior and inferior grains was inhibited; CT significantly increased the soil moisture and thus promoted the filling of both the inferior and superior grains. 4.2. Relationship of hormone changes and wheat grain filling Cytokinins (CTKs) play an important role in regulating grain filling, and it has been reported that 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 the endosperm of developing seeds and may be required for cell division during the early phase of seed setting (Michael and SeilerKelbitsch, 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 (Xu et al., 2007; Yang and Zhang, 2006; Zhang et al., 2009a,c). The present study indicated that the IAA and Z + ZR contents in the grains were positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates (Table 4), indicating that Z + ZR and IAA are involved in regulating wheat grain filling. In addition, the changes in the grain IAA and Z + ZR contents presented similar patterns of change. The Z + ZR and IAA contents in the grains transiently increased in the early grain-filling stage and then decreased, reaching a maximum at 12 days post-anthesis for the superior grains and 15 days postanthesis for the inferior grains. We also observed that the maximal IAA and Z + ZR contents occurred just before the maximal grainfilling rate in both the superior and inferior grains. Xu et al. (2007) suggested that CTKs regulate endosperm cell division in developing rice grains, and Davies (1987) stated that auxin also stimulates cell division. High IAA levels in a sink organ can create an “attractive power”, leading to increased cytokinin levels in grains (Seth and Waering, 1967; Singh and Gerung, 1982). These results suggest that IAA and Z + ZR may regulate wheat grain filling in the early filling stage, most likely via the manipulation of endosperm cell division, thereby creating sink strength. In addition to Z + ZR and IAA, ABA and ETH also play important roles in regulated grain filling. Yang et al. (2006) suggested that the higher ABA concentration and lower ETH concentration found in superior versus inferior wheat grains were associated with a higher filling rate in the superior grains. Our present study found a similar result. The ABA content in the superior grains was significantly

higher than in the inferior grains during the early grain-filling stage (3–15 days post-anthesis); however, the ETH content in the superior grains was significantly lower than that in the inferior grains during the grain-filling stage. Our regression analysis indicated that the ABA content in the grains was positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. In contrast, the ETH content in the grains was negatively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. These results indicate that ABA promotes wheat grain filling and that ETH inhibits wheat grain filling, findings that are consistent with a previous study (Yang et al., 2006). There are some reports that GAs are also involved in regulating grain development. Eeuwens and Schwabe (1975) have stated that a GA-like material is at the highest level in the liquid endosperm of peas during rapid pod elongation. Relatively high levels of GA1 ,4 ,19 exist in the large panicles of rice just before and at anthesis (Kurogochi et al., 1979; Suzuki et al., 1981). In the present study, the GAs in the superior grains were significantly lower than in the inferior grains; however, the GA content in the grains was not significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. These results suggest that GAs may be involved in the regulation of wheat grain filling but may not be a determining factor for this process in wheat. In the present study, CT significantly increased the IAA and Z + ZR contents and decreased the ABA, ETH, and GA contents in the inferior grains at the 220- and 260-mm rainfall levels; at the 220-mm rainfall level, CT significantly increased the IAA and Z + ZR contents and decreased the ABA, ETH, and GA contents in the superior grains. A regression analysis indicated that the ratios of IAA/ETH, Z + ZR/ETH, and ABA/ETH were all positively and significantly correlated with the maximum grain weight and the maximum and mean grain-filling rates. These results indicate that the balance of hormones, rather than the individual hormone content, regulated the grain filling of wheat; thus, CT, through the regulation of the balance of hormones, affected the grain filling of wheat. Further studies on the effect of individual hormone levels and the interaction of hormones on the regulation of wheat grain weight are necessary.

5. Conclusions The effect of CT on wheat grain filling is significantly related to precipitation levels. At the 220-mm rainfall level, CT significantly promoted the filling of superior and inferior grains. At the 260-mm rainfall level, CT also significantly promoted the filling of inferior grains yet had no significant effect on the filling of superior grains. In contrast, CT had no significant effect on the grain filling rates and grain weight of superior and inferior grains at the 300-mm rainfall level. At 220- and 260-mm rainfall, CT significantly increased the IAA and Z + ZR contents and decreased the ABA, ETH, and GA contents in inferior grains. CT also significantly increased the IAA and Z + ZR contents and decreased the ABA, ETH, and GA contents in superior grains at 220-mm rainfall. In addition, the precipitation level and CT both significantly increased the soil moisture during the grain-filling stage. Based on these results, we conclude that CT significantly increased the soil moisture, thereby regulating wheat grain filling, a process that was significantly related to the balance of hormones in the analyzed grains.

Acknowledgement This work was supported by the National Natural Science Foundation of China (Project No. 30971721, 31070375, 31171506).

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