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High nitrogen input causes poor grain filling of spikelets at the panicle base of super hybrid rice
Field Crops Research 244 (2019) 107635
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High nitrogen input causes poor grain filling of spikelets at the panicle base of super hybrid rice Penghao Fua, Jing Wanga, Tong Zhanga, Jianliang Huanga,b, Shaobing Penga,
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a National Key Laboratory of Crop Genetic Improvement, MARA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China b Hubei Collaborative Innovation Center for Grain Industry, Yangtze University, Hubei, 434023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Grain filling High nitrogen input Large-panicle cultivar Rice Spikelet position
It is generally accepted that poor grain filling of rice plants under high nitrogen (N) input is due to increased competition for assimilates because high N rate usually results in large sink size with more spikelets per panicle. In this study, we examined grain filling parameters of spikelets in different panicle position under various N treatments to determine the effect of N rates on grain filling. A super rice cultivar with large panicle was grown under field conditions in Wuxue County, Hubei Province, central China in 2013 and 2014. Fully filled spikelet percentage measured at the crop level from 12 hills decreased by 10.9% when the total N rate increased from 0 to 250 kg ha−1, whereas spikelets per panicle remained almost constant across the N rates. At the individual panicle level, reduction in grain filling was evidenced by decreased fully filled spikelet percentage, fertilized spikelet weight, and grain plumpness, and increased partially filled spikelet percentage in the bottom part of the panicle as total N rates increased. Reduction in these grain filling parameters under high N input was relatively small in the middle part of the panicle and was not appeared in the top part of the panicle. Furthermore, high N rates at both mid-tillering and panicle initiation reduced fertilized spikelet weight in the bottom part of the panicle without an increase in spikelets per panicle. Our results suggest that the spikelets in the bottom part of the panicle were mainly responsible for poor grain filling under high N input and such high N-induced poor grain filling was not due to increased spikelets per panicle.
1. Introduction Rice (Oryza sativa L.) is one of the world’s most important crops and the foremost staple food in Asia (GRiSP, 2013). In China, rice accounts for one-third of the total grain production, which is important for national food security (Xie, 2007). China has successfully bred and released many super rice cultivars to meet the food demands of the rapidly growing population (Cheng et al., 2007). Most of these cultivars have the common characteristics of large panicles (large number of spikelets per panicle), which is one of the most important contributors to high grain yield (Sheehy et al., 2001). However, super rice cultivars do not always meet the expectation in yield performance due to poor grain filling (Huang et al., 2008). The degree of grain filling in rice spikelets can be characterized by the percentage of spikelet filling, the final weight of filled spikelets, and grain plumpness. The degree and rate of grain filling in rice spikelets differ largely with their positions on a panicle (Zhang et al., 2010). Based on their flowering date and position within a panicle, the
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spikelets can be classified as superior and inferior spikelets (Zhu et al., 1988). Superior spikelets flower earlier and are generally located on the top part of the panicle, while inferior spikelets flower later and are generally located on the bottom part of the panicle (Yang et al., 2006). Superior spikelets usually exhibit greater filling rate and heavier final weight than inferior ones (Zhang et al., 2010). Yang and Zhang (2010) reported that the poor grain-filling problem in inferior spikelets is more aggravated in the super rice cultivars. Nitrogen (N) is one of the most important nutrients for the growth and development of rice plants. It can significantly increase grain yield by expanding source and sink capacity (Ding et al., 2014; Fageria and Santos, 2015). However, a higher amount of N than the minimum required to maintain maximum crop growth is often applied to maximize rice grain yield (Lemaire and Gastal, 1997). This problem is more common in China because the average rate of N-fertilizer application for rice in 2006 was 193 kg ha−1, which is approximately 90% higher than the world average (Peng et al., 2010). Over application of N fertilizer is more severe when super rice cultivars are planted because
Corresponding author. E-mail address: [email protected] (S. Peng).
https://doi.org/10.1016/j.fcr.2019.107635 Received 25 July 2019; Received in revised form 22 September 2019; Accepted 23 September 2019 0378-4290/ © 2019 Published by Elsevier B.V.
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lodging does not happen easily with super rice cultivars (Wang et al., 2002b). Peng et al. (2010) reported that there was no positive correlation between grain yield and the amount of N applied based on a large dataset across locations and years in China. Yield reduction is often observed when the high rates of N fertilizer are applied even for the super rice cultivars (Tang, 2003; Liang et al., 2015). A decrease in grain yield under excessive N application was usually attributed to increased susceptibility to lodging (Duy et al., 2004), damage from pests and diseases (Wu et al., 2012), and mutual shading (Tanaka, 1966). Reduction in rice grain filling under high N input was observed in several studies (Ohnishi et al., 1999; Yang et al., 2001b; Jiang et al., 2016), which could be the most direct explanation for the decreased grain yield caused by excessive N application. However, researchers seldom attribute high-N induced yield decline to poor grain filling because high N input can also increase spikelets per panicle (Kamiji et al., 2011; Ding et al., 2014). The degree of grain filling can be reduced under increased sink size due to competition for assimilates (Li et al., 2013). For example, Jiang et al. (2016) reported that spikelet filling decreased by 7 percentage points while spikelets per panicle increased by 25% when N rate at panicle initiation (PI) increased from zero to 180 kg ha−1. In this case, the effect of high N rate on grain filling was confounded by the increased spikelets per panicle. Therefore, the effect of high N input on grain filling should be determined when sink size defined as spikelets per panicle is not increased significantly by high N rates. In this study, the effect of N input on the grain filling of a super rice cultivar was examined when N rates did not affected spikelets per panicle significantly. The objectives of this study were (1) to determine the effect of N rates at different growth stage on the grain filling of a super rice cultivar with large panicles; and (2) to compare spikelets of different panicle position in their grain filling under high N rates.
Table 1 Nitrogen treatments and their application rates (kg ha−1) as basal and topdressing at mid-tillering (MT) and panicle initiation (PI). N treatments
Basal
MT
PI
Total N
0-0-0 0-0-40 0-0-80 90-0-0 90-0-40 90-0-80 90-40-0 90-40-40 90-40-80 90-80-0 90-80-40 90-80-80
0 0 0 90 90 90 90 90 90 90 90 90
0 0 0 0 0 0 40 40 40 80 80 80
0 40 80 0 40 80 0 40 80 0 40 80
0 40 80 90 130 170 130 170 210 170 210 250
Guangzhan63-4S as the female parent and Yangdao 6 as the male parent (Dai et al., 2005). YLY6 was released by the ministry of agriculture as a super hybrid rice cultivar in 2009. The seedlings were raised in a seedbed with a sowing date of May 12 for both years. The soil was plowed and puddled before transplantation. Transplanting was done on June 12, 2013 and June 16, 2014 at a density of 25 hills m−2 and a hill spacing of 13.3 × 30.0 cm with two seedlings per hill. Phosphorus (40 kg P ha-1 as calcium superphosphate) and zinc (5 kg Zn ha-1 as zinc sulfate heptahydrate) were applied at 1 d before transplanting. Potassium (200 kg K ha-1 as potassium chloride) was split equally and applied at 1 d before transplanting and at PI. To minimize leakage between plots, all bunds were covered with plastic film and the plastic film was installed to a depth of 30 cm below soil surface. The paddy fields were flooded after transplanting, and a floodwater depth of 3–5 cm was maintained until 1 week prior to harvest except that the water was drained at maximum tillering stage for about 1 week to reduce unproductive tillers. To prevent biomass and yield losses, pests, diseases, and weeds were intensively controlled throughout the rice growth period in both years.
2. Materials and methods 2.1. Site description Experiments were conducted in farmers’ fields at Lanjie Village, Huaqiao Township, Wuxue County, Hubei Province, China (30°00′ N, 115°44′ E) during the rice-growing season from May to October in 2013 and repeated in an adjacent field in 2014. Soil samples from the upper 20 cm layer were collected for soil analysis before basal fertilizer was applied in both years. The average values of soil properties for the experimental plots across the two years were as follows: a pH of 5.6, organic matter of 32.9 g kg−1, total N of 2.48 g kg−1, Olsen-P of 9.38 mg kg−1, and available K of 112.9 mg kg−1. In both years, climate data were collected during the growing period from transplanting to maturity from a weather station (AWS 800, Campbell Scientific, Inc., USA), located about 100-meter distance from the experimental field. Seasonal average daily minimum temperature, maximum temperature, and solar radiation were 24.6 °C, 32.2 °C, and 17.7 MJ m-2, respectively in 2013 and 23.0 °C, 30.1 °C, and 14.4 MJ m-2, respectively in 2014.
2.3. Sampling and measurement At heading, 100 panicles with similar development stage were labeled on the same day in each plot. Six labeled panicles per plot were sampled at 3-day intervals from heading to maturity. Based on panicle length, the panicles were divided into three equal parts: top, middle, and bottom. Each part was immediately dried in an oven at 80 °C to cease its metabolic activity. Then, all spikelets were detached from panicles by hands. For spikelets sampled at heading, spikelet projection area of 100 spikelets per plot was measured using an Epson Perfection V700 Photo scanner at 300 DPI resolution. The photographs were analyzed using ImageJ 1.6.0 software to determine the length, width, and area of the spikelet. Starting at 3 d after heading, partially filled spikelets were separated from unfilled spikelets by visual observation plus pinching with fingers. For the final samples at maturity, all spikelets without unfilled ones were submerged in a sodium chloride solution with the specific gravity of 1.10 to separate high density grains from others. The floating samples were then submerged in tap water to separate fully filled spikelets from partially filled ones. Spikelet numbers of each category from the top, middle, and bottom parts of the panicle were counted and then samples were dried in an oven at 80 °C to constant weight. In this paper, we divided spikelets into high density grains, fully filled spikelets (including high density grains), partially filled spikelets, and unfilled spikelets. For convenience, all spikelets except for unfilled ones were referred to fertilized spikelets in this study. Grain plumpness was calculated as percent of the dry weight of fertilized spikelets to that of high density grains (Zhu et al., 1995). Because high density grain weights varied across panicle position, the grain plumpness of top,
2.2. Experimental design and crop management The experiments with twelve N treatments were laid out in a completely randomized block design with four replications. Plot size was 35.75 m2 (5.5 × 6.5 m). The twelve N treatments were consisted of various rates and timing of N applied as basal fertilizer (manually broadcast and incorporated in plots 1 d before transplanting) and as topdressing at mid-tillering (MT) and PI. Two N rates with 0 and 90 kg ha−1 were applied as basal and three N rates with 0, 40, and 80 kg ha−1 were applied at MT or PI. The total amount of N application ranged from 0 to 250 kg N ha−1. All N fertilizers were applied in the form of urea. Detailed information about N treatments was listed in Table 1. A widely grown rice cultivar in central China, Yangliangyou 6 (YLY6) with large panicles, was used as the experimental material. YLY6 is an indica hybrid cultivar developed by two-line system in 2001 with 2
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Table 2 Analysis of variance for grain yield and yield components. Source
Grain yield
Panicles m−2
Spikelets per panicle
Spikelets m−2
Fully filled spikelet %
Fully filled spikelet wt
Year N Year × N
* ** ns
** ** **
ns ns ns
ns ** ns
* ** ns
** ** ns
* and **, significant at the 0.05 and 0.01 probability levels, respectively; ns, not significant at 0.05 probability level.
Fig. 1. Responses of grain yield and yield components to total nitrogen rates. Grain yield and yield components were determined at maturity based on the samples of 5 m2 and 12 hills per plot, respectively. Data were averaged across the two years. Error bars indicate ± SE (n = 4).
oven-dried at 80 °C to constant weight. Spikelets per panicle (total spikelets m-2 / panicles m−2) and fully filled spikelet percentage (100 ×fully filled spikelets m−2 / total spikelets m−2) were calculated. Source-sink ratio was calculated as the ratios of dry matter accumulation (DMA) at heading, maturity, and between heading and maturity to the product of total spikelets m−2 and the weight of high density grain. Yield was determined from a 5-m2 area at maturity in each plot and adjusted to the standard moisture content of 0.14 g H2O g-1 fresh weight. Grain moisture content was measured with a digital moisture tester (DMC-700, Seedburo, Chicago, IL, USA). Plant N concentration of the samples for yield component was determined by an elemental analyzer (Elementar vario MAX CNS/CN, Elementar Trading Co., Ltd, Germany). Total N uptake was the summation of N content of straw, fully filled spikelets, and the combined sample of rachis, partially filled spikelets, and unfilled spikelets.
middle, and bottom parts of the panicle was calculated based on the high density grain weights of the three parts of the panicle, respectively. Twelve hills were sampled from each plot at maturity to measure yield components. Plant samples were separated into straw and panicle after recording panicle numbers. Dry weight of straw was determined after oven-dried at 80 °C to constant weight. Panicles were hand-threshed and fully filled spikelets were separated from others by submerging them in tap water. Unfilled spikelets were separated from partially filled ones by winnowing. Three subsamples with each of 30 g fully filled spikelets and 2 g unfilled spikelets were taken to determine the numbers of fully filled and unfilled spikelets, whereas the entire sample was counted to determine the number of partially filled spikelets. The numbers of fully filled, partially filled, and unfilled spikelets were added to determine total spikelets m−2. Dry weights of rachis, fully filled, partially filled, and unfilled spikelets were determined after 3
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Fig. 2. Responses of fertilized, fully filled, and partially filled spikelet percentages of the top (T), middle (M), and bottom (B) parts of the panicle to total nitrogen rates in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
did not result in an increase in grain yield (Fig. 1a, Table S1). The yield response to the N rate was mainly explained by the difference in spikelets m−2 across N treatments (Fig. 1b), which was determined primarily by panicles m−2 (Fig. 1c), not by spikelets per panicle (Fig. 1d). In fact, spikelets per panicle remained almost constant across the N rates. In contrast, there was a declining trend in fully filled spikelet percentage with the increase in total N rate (Fig. 1e). Fully filled spikelet percentage decreased by 10.9% when the total N rate increased from 0 to 250 kg ha-1 (Table S1). A slight increase in fully filled spikelet weight was observed when the total N rate increased from 0 to 90 kg ha1 , and further increase of the N rate did not affect this trait significantly (Fig. 1f). The responses of DMA, tissue N concentration, and total N uptake were similar between 2013 and 2014 since the interactive effects between year and N treatments were insignificant for these traits (Table S2). Dry matter accumulation (DMA) at heading and maturity increased with the increase in total N rate, whereas DMA between heading and maturity remained relatively stable across the N treatments (Fig. S1a-c, Table S3). Nitrogen concentrations of straw and fully filled spikelets increased with the increase in total N rate (Fig. S3a-b, Table S3). This was also true for total N uptake (Fig. S3c, Table S3). Fertilized spikelet percentage remained fairly constant across total
2.4. Statistical analysis Statistical data analysis was performed using analysis of variance (Statistix 9.0, Analytical Software, Tallahassee, FL, USA). Year, N management, and panicle position were considered fixed effects and block was considered a random effect. When significant, the mean values were compared by the least significance difference (LSD) test at the 0.05 probability level. For variables without significant year × N treatment interactions, data were presented with means across the two years. The responses of agronomic and grain filling traits to total N rates were fitted with linear or quadratic functions, while the time-course of fertilized spikelet weight was fitted with Richards’ growth equation (Richards, 1959). 3. Results Grain yield and yield components except for spikelets per panicle were significantly affected by the N treatments, whereas the interactive effects between year and N treatments were insignificant for grain yield and yield components except for panicles m−2 (Table 2). Average grain yield across the two years increased with the increase in total N rate until the N rate reached 210 kg ha-1 and further increase of the N rate 4
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Fig. 3. Responses of fully filled and fertilized spikelet weights of the top (T), middle (M), and bottom (B) parts of the panicle to total nitrogen rates in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
rates and years, fertilized spikelet weight of the bottom part of the panicle was 17.5% lower than that of the top part of the panicle. Figs. 4 and 5 showed the time-course of grain development from heading to maturity for the three N rates of 0, 40, and 80 kg ha−1 applied at MT and PI, respectively. In the top part of the panicle, zero N rate at MT had higher fertilized spikelet weight than the N rates of 40 and 80 kg ha−1 at MT during the early phase of grain development, and the difference disappeared when fertilized spikelet weight reached maximum level (Fig. 4a, d). Similar response of fertilized spikelet weight to the N rate at MT was observed in the middle part of the panicle except for the late phase of grain development in 2013 when the N rates of 0 and 40 kg ha−1 at MT had higher fertilized spikelet weight than the N rate of 80 kg ha−1 at MT (Fig. 4b, e). In the bottom part of the panicle, the three N rates had similar fertilized spikelet weight up to 12 d after heading, and since then the high N rate at MT reduced fertilized spikelet weight significantly, especially in the late phase of grain development (Fig. 4c, f). The N rates at PI had smaller effect on grain filling during the early phase of grain development than that at MT in the top and middle parts of the panicle (Figs. 4 and 5). Fertilized spikelet weight was not affected by the N rate at PI in the top part of the panicle in both years and in the middle part of the panicle in 2014 (Fig. 5a, d, e). The N application at PI reduced fertilized spikelet weight significantly in the late phase of grain development in the middle part of the panicle in 2013 (Fig. 5b). In the bottom part of the panicle, the N rates at PI had a similar effect on fertilized spikelet weight as the N rates at MT (Fig. 5c, f). Therefore, excessive N application at MT and PI had significantly negative effect on the grain filling of the bottom part of the panicle. Grain plumpness decreased with the increase in total N rate in the bottom and middle parts of the panicle, whereas the grain plumpness of the top part of the panicle remained relatively constant across total N rates (Fig. 6a-b). The grain plumpness of the top part of the panicle was maintained at more than 94% regardless of the N rates, whereas grain plumpness of the middle and bottom parts of the panicle reduced to as low as 85.7% and 64.4%, respectively, when the total N rate increased from 0 to 250 kg ha−1. Difference between the top and middle parts of
N rates, especially in 2013 (Figs. 2a, d), this was evidenced by the insignificant effect of the N treatment on this trait in 2013 (Table S4). The bottom part of the panicle had lower fertilized spikelet percentage than the top and middle parts of the panicle. Such difference was more consistent in 2013 than 2014. There was no difference in fertilized spikelet percentage between the top and middle parts of the panicle in 2013, and the difference was significant only at the N rate of 250 kg ha−1 in 2014. Fully filled spikelet percentage decreased while partially filled spikelet percentage increased with the increase in total N rate except for the top part of the panicle in 2013 (Fig. 2b-c, e-f). This was supported by the fact that the interactive effects between the N treatment and panicle position were significant for both fully and partially filled spikelet percentages (Table S4). In general, the bottom part of the panicle showed the greatest responses to high N input in fully and partially filled spikelet percentage, followed by the middle and top parts of the panicle. The bottom part of the panicle had lower fully filled spikelet percentage and higher partially filled spikelet percentage than the top and middle parts of the panicle across N treatments. There was no difference in fully filled spikelet percentage and partially filled spikelet percentage between the top and middle parts of the panicle under low N conditions, but the difference was significant under high N conditions. Averaged across the N rates and years, fully filled spikelet percentage of the top part of the panicle was 56.5% higher than that of the bottom part of the panicle, while partially filled spikelet percentage in the top part of the panicle was only 20.9% of that of the bottom part of the panicle. Fully filled spikelet weight showed a slightly increasing trend with the increase in total N rate and the top part of the panicle had the highest fully filled spikelet weight, followed by the middle and bottom parts of the panicle (Fig. 3a, c). Fertilized spikelet weight decreased with the increase in total N rate in the middle and bottom parts of the panicle in 2013 and only in the bottom part of the panicle in 2014 (Fig. 3b, d). The bottom part of the panicle had lower fertilized spikelet weight than the top and middle parts of the panicle. Difference between the top and middle parts of the panicle in fertilized spikelet weight was significant only at the N rate of 250 kg ha−1. Averaged across the N 5
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Fig. 4. Changes in fertilized spikelet weights of the top (T), middle (M), and bottom (B) parts of the panicle during grain filling period for the nitrogen rates of 0, 40, and 80 kg N ha−1 applied at mid-tillering in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
maturity did not decrease across the N rates (Fig. S1c), and (3) reduction in source-sink ratios calculated at heading, maturity, and between heading and maturity was not observed consistently across the two years (Fig. S2). Furthermore, the poor grain filling under high N conditions was not due to the N deficiency of rice crop because high N concentrations of straw and fully filled spikelets and high total N uptake were observed under high N input (Fig. S3). At the individual panicle level, reduction in grain filling was evidenced by decreased fully filled spikelet percentage, fertilized spikelet weight, and grain plumpness, and increased partially filled spikelet percentage in the bottom part of the panicle as total N rate increased (Figs. 2,3, and 6). Reduction in these grain filling parameters under high N input was relatively small in the middle part of the panicle and was not appeared in the top part of the panicle. The reduction in fully filled spikelet percentage and grain plumpness in the bottom part of the panicle was unlikely due to increased spikelet size under high N input because spikelet area remained stable across the N rates for all three parts of the panicle (Fig. S4). Furthermore, fertilized spikelet weight during grain-filling period was compared among the three N rates (0, 40, and 80 kg ha−1) at MT and PI. High N rates at both MT and PI reduced fertilized spikelet weight in the bottom part of the panicle (Figs. 4 and 5). Again, high N rates at both MT and PI did not increase
the panicle in grain plumpness was significant only at the N rate of 250 kg ha−1. 4. Discussion Reduction in grain filling under high N input was observed in this study at both crop and individual panicle levels. More importantly, the reduced grain filling was not due to increased sink size because spikelets per panicle remained relatively stable across the N rates. At the crop level, fully filled spikelet percentage decreased by 10.9% when the total N rate increased from 0 to 250 kg ha−1, while spikelets per panicle did not increase across the N rates based on the data from 12-hill samples (Fig. 1). Fully filled spikelet weight did not decrease significantly when the total N rate increased from 130 to 250 kg ha−1. These results suggest that the stagnation of rice grain yield under high N conditions was mainly caused by poor grain filling. This result is in consistent with Jiang et al. (2016) who reported reduction in grain filling under the highest N rate of 180 kg ha-1 applied at PI. The poor grain filling under high N conditions was unlikely due to increased competition for assimilates because of the following reasons: (1) dry matter accumulation (DMA) at heading and maturity increased with the increase in total N rate (Fig. S1a-b), (2) DMA between heading and 6
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Fig. 5. Changes in fertilized spikelet weights of the top (T), middle (M), and bottom (B) parts of the panicle during grain filling period for nitrogen rates of 0, 40, and 80 kg N ha−1 applied at panicle initiation in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
Fig. 6. Responses of grain plumpness of the top (T), middle (M), and bottom (B) parts of the panicle to total nitrogen rates in 2013 and 2014. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
rice plants (Yao et al., 2000; Fukushima et al., 2011; Jiang et al., 2016). High N application at vegetative stage increases spikelets m−2 by promoting tillering and panicles m−2 (Sui et al., 2013), while high N application at PI increases sink size by enhancing spikelets per panicle
spikelets per panicle (Table S5), suggesting that the reduced fertilized spikelet weight in the bottom part of the panicle under high N input was not due to increased sink size. Increased sink size under high N input was commonly observed in 7
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(Ding et al., 2014). In our study, spikelets m−2 reached almost maximum level when total N rate increased to 130 kg ha-1 and spikelets per panicle remained stable across all N rates. The lack of response of sink size to high N input might be due to high indigenous N supply of the soil in our study which was evidenced by the fact that over 7 t ha-1 of grain yield was produced without N application. Wang et al. (2002a) reported that the spikelets per panicle of tiller stems began to decrease sharply when the N concentration in the top two leaves increased to a certain level, which might explain the lack of response of sink size to increased N rates. High N input also resulted in high N accumulation and enhanced N metabolism in rice plant and thereby delayed and decreased non-structural carbohydrate accumulation, which could reduce spikelets per panicle (Li et al., 2010). Our study indicates that the spikelets in the bottom part of the panicle were mainly responsible for reduced grain filling under high N input. Poor grain filling of spikelets in the bottom part of the panicle has been reported for cultivars with large panicles such as intersubspecific hybrid between indica and japonica (Liang et al., 2001). These spikelets are considered as inferior ones because they usually exhibit lower filling rate and lighter final weight than superior ones (Yang et al., 2006). Over the last few decades, considerable effort has been made to elucidate the mechanisms underlying poor grain filling of spikelets in the bottom part of the panicle and to identify a solution for this problem. It has been suspected that the poor grain filling of inferior grains may be due to the lack of assimilate supply (Li et al., 2013), weak sink strength (Liang et al., 2001), endogenous hormone inhibition (Wang et al., 2006), low activity of key enzymes associated with sucrose–starch metabolism (Yang et al., 2001a), and poor transport capacity (Huang et al., 2005). The precise mechanism and strategies that could be used to enhance grain filling remain unclear (Yang and Zhang, 2010). In the present study, we demonstrated that poor grain filling of the bottom part of the panicle became a limiting factor for further increase in rice grain yield under high N treatments. As stated above, poor grain filling of the basal part of the panicle under high N application rates was not attributed to reduction in source-sink ratio, N deficiency, increased sink size, or increased spikelet size. Previous studies reported that ethylene had an adverse effect on the grain filling of rice, especially for cultivars with large panicles because of the longer period of exposure of the lower part of the large panicles to the gaseous ethylene (Mohapatra and Panigrahi, 2011; Yang and Zhang, 2005). Ethylene inhibited the partitioning of assimilate in developing spikelets and resulted in low starch biosynthesis and high accumulation of soluble carbohydrate (Sekhar et al., 2015). The spikelets in the bottom part of the panicle initiated earlier but flowered later than the spikelets in the top part of the panicle, which meant longer exposure in ethylene environment for the spikelets in the bottom part of the panicle (Ikeda et al., 2004). High N input might aggravate the ethylene effect on spikelets in the bottom part of the panicle through prolonging the ethylene exposure duration, because high N input always cause longer panicle development duration (Liu et al., 2005). Yang and Zhang (2005) stated that when plant senescence is unfavorably delayed under high N conditions, rice will exhibit prolonged and slow grain filling. High N application was accompanied by an increased tiller number and leaf area, which led to mutual shading, canopy closure and reduced light penetration into the rice canopy (Tanaka, 1966). Excessive N concentrations in plant tissue might result in a high rate of N metabolism and overconsumption of carbohydrate, thereby reducing carbohydrate supply for grain filling (Li et al., 2010; Liang et al., 2015). Given that carbohydrate is the main composition of endosperm, the carbohydrate acceptance capacity of the endosperm may also play a vital role in grain filling (Wang et al., 2012). The cell number, size, and filling degree of endosperm are all closely related to grain weight and grain filling (Yang et al., 2006; Li et al., 2013). Nevertheless, the physiological mechanism underlying poor grain filling of the bottom part of the panicle under high N input should be further studied in future.
5. Conclusions This study showed that high N input induced poor grain filling of spikelets at the panicle base. Fully filled spikelet percentage, fertilized spikelet weight, and grain plumpness decreased, while partially filled spikelet percentage increased in the bottom part of the panicle as total N rates increased. The high N-induced poor grain filling observed in this study was not due to increased sink size because spikelets per panicle remained relatively stable across the N rates. Our results suggest that high N input could reduce the grain filling of rice crop without the increased spikelets per panicle. Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0300210) and a grant from the National Natural Science Foundation of China (No. 31671620). We acknowledge the assistance from staff members of the Modern Agricultural Demonstration Center of Wuxue County. References Cheng, S., Cao, L., Zhuang, J., Chen, S., Zhan, X., Fan, Y., Zhu, D., Min, S., 2007. Super hybrid rice breeding in China: achievements and prospects. J. Integr. Plant Biol. 49, 805–810. Dai, Z., Liu, G., Li, A., Xu, M., Liu, X., Zhou, C., Zhang, H., 2005. Breeding of two-line indica hybrid rice combination, "Yangliangyou 6″, and studying on its culture characteristics. Chin. Agric. Sci. Bull. 21, 114–116. Ding, C., You, J., Chen, L., Wang, S., Ding, Y., 2014. Nitrogen fertilizer increases spikelet number per panicle by enhancing cytokinin synthesis in rice. Plant Cell Rep. 33, 363–371. Duy, P.Q., Abe, A., Hirano, M., Sagawa, S., Kuroda, E., 2004. Analysis of lodging-resistant characteristics of different rice genotypes grown under the standard and nitrogen-free basal dressing accompanied with sparse planting density practices. Plant Prod. Sci. 7, 243–251. Fageria, N.K., Santos, A.B., 2015. Yield and yield components of lowland rice genotypes as influenced by nitrogen fertilization. Commun. Soil Sci. Plant Anal. 46, 1723–1735. Fukushima, A., Shiratsuchi, H., Yamaguchi, H., Fukuda, A., 2011. Effects of nitrogen application and planting density on morphological traits, dry matter production and yield of large grain type rice variety bekoaoba and strategies for super high-yielding rice in the Tohoku region of Japan. Plant Prod. Sci. 14, 56–63. GRiSP (Global Rice Science Partnership), 2013. Rice Almanac, 4th edition. International Rice Research Institute, Los Baños, the Philippines 283 p. Huang, M., Mo, R., Zou, Y., Mo, Y., Zhan, K., Jiang, P., 2008. Yield components and grain filling characters of super hybrid rice. Crop Res. 22, 249–253. Huang, S., Zou, Y., Liu, C., 2005. Setting physiology of the superior and inferior grains of hybrid rice Liangyoupeijiu. Acta Agron. Sin. 1, 102–107. Ikeda, K., Sunohara, H., Nagato, Y., 2004. Developmental course of inflorescence and spikelet in rice. Breed. Sci. 54, 147–156. Jiang, Q., Du, Y., Tian, X., Wang, Q., Xiong, R., Xu, G., Yan, C., Ding, Y., 2016. Effect of panicle nitrogen on grain filling characteristics of high-yielding rice cultivars. Eur. J. Agron. 74, 185–192. Kamiji, Y., Yoshida, H., Palta, J.A., Sakuratani, T., Shiraiwa, T., 2011. N applications that increase plant N during panicle development are highly effective in increasing spikelet number in rice. Field Crops Res. 122, 242–247. Lemaire, G., Gastal, F., 1997. N uptake and distribution in plant canopies. In: Lemaire, G. (Ed.), Diagnosis of the Nitrogen Status in Crops. Springer, Berlin Heidelberg, Berlin, Heidelberg, pp. 3–43. Li, G., Wang, H., Wang, S., Wang, Q., Zheng, Y., Ding, Y., 2010. Effect of nitrogen applied at rice panicle initiation stage on carbon and nitrogen metabolism and spikelets per panicle. J. Nanjing Agric. Univ. 1, 1–5. Li, X., Cheng, H., Wang, N., Yu, C., Qu, L., Cao, P., Hu, N., Liu, T., Lyu, W., 2013. Critical factors for grain filling of erect panicle type japonica rice cultivars. Agron. J. 105, 1404–1410. Liang, J., Zhang, J., Cao, X., 2001. Grain sink strength may be related to the poor grain filling of indica-japonica rice (Oryza sativa L.) hybrids. Physiol. Planta. 112, 470–477. Liang, Z., Bao, A., Li, H., Cai, H., 2015. The effect of nitrogen level on rice growth, carbonnitrogen metabolism and gene expression. Biologia 70, 1340–1350. Liu, X.W., Meng, Y.L., Zhou, Z.G., Cao, W.X., 2005. Dynamic characteristics of floret differentiation and degeneration in rice. Acta Agron. Sin. 314, 451–455. Mohapatra, P., Panigrahi, R., 2011. Ethylene control of grain development in the inferior spikelets of rice panicle. Adv. Plant Physiol. 12, 79–89. Ohnishi, M., Horie, T., Homma, K., Supapoj, N., Takano, H., Yamamoto, S., 1999. Nitrogen management and cultivar effects on rice yield and nitrogen use efficiency in
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development and its mineral element analysis. Chin. J. Rice Sci. 26, 693–705. Wu, W., Huang, J., Cui, K., Nie, L., Wang, Q., Yang, F., Shah, F., Yao, F., Peng, S., 2012. Sheath blight reduces stem breaking resistance and increases lodging susceptibility of rice plants. Field Crops Res. 128, 101–108. Xie, H., 2007. Advances on the breeding program and technology research in south China type super-rice. J. Shenyang Agric. Univ. 5, 714–718. Yang, J., Peng, S., Gu, S., Visperas, R., Zhu, Q., 2001a. Changes in activities of three enzymes associated with starch synthesis in rice grains during grain filling. Acta Agron. Sin. 2, 157–164. Yang, J., Zhang, J., Wang, Z., Zhu, Q., Wang, W., 2001b. Hormonal changes in the grains of rice subjected to water stress during grain filling. Plant Physiol. 127, 315–323. Yang, J., Zhang, J., 2005. Grain filling of cereals under soil drying. New Phytol. 169, 223–236. Yang, J., Zhang, J., Wang, Z., Liu, K., Wang, P., 2006. Post-anthesis development of inferior and superior spikelets in rice in relation to abscisic acid and ethylene. J. Exp. Bot. 57, 149–160. Yang, J., Zhang, J., 2010. Grain-filling problem in “super” rice. J. Exp. Bot. 61, 1–4. Yao, Y., Yamamoto, Y., Wang, Y., Yoshida, T., Miyazaki, A., Nitta, Y., Cai, J., 2000. Role of nitrogen regulation in sink and source formation of high-yielding rice cultivars. Soil Sci. Plant Nutr. 46, 825–834. Zhang, H., Chen, T., Wang, Z., Yang, J., Zhang, J., 2010. Involvement of cytokinins in the grain filling of rice under alternate wetting and drying irrigation. J. Exp. Bot. 61, 3719–3733. Zhu, Q., Cao, X., Luo, Y., 1988. Growth analysis on the process of grain filling in rice. Acta Agron. Sin. 3, 182–193. Zhu, Q., Wang, Z., Zhang, Z., Hui, D., 1995. Study on indicators of grain-filling of rice. J. Jiangsu Agric. Coll. 16, 1–4.
Northeast Thailand. Field Crops Res. 64, 109–120. Peng, S., Buresh, R.J., Huang, J., Zhong, X., Zou, Y., Yang, J., Wang, G., Liu, Y., Hu, R., Tang, Q., 2010. Improving nitrogen fertilization in rice by site-specific N management: a review. Agron. Sustain. Dev. 30, 649–656. Richards, F.J., 1959. A flexible growth function for empirical use. J. Exp. Bot. 10, 290–301. Sekhar, S., Panda, B.B., Mohapatra, T., Das, K., Shaw, B.P., Kariali, E., Mohapatra, P.K., 2015. Spikelet-specific variation in ethylene production and constitutive expression of ethylene receptors and signal transducers during grain filling of compact- and laxpanicle rice (Oryza sativa) cultivars. J. Plant Physiol. 179, 21–34. Sheehy, J.E., Dionora, M.J.A., Mitchell, P.L., 2001. Spikelet numbers, sink size and potential yield in rice. Field Crops Res. 71, 77–85. Sui, B., Feng, X., Tian, G., Hu, X., Shen, Q., Guo, S., 2013. Optimizing nitrogen supply increases rice yield and nitrogen use efficiency by regulating yield formation factors. Field Crops Res. 150, 99–107. Tanaka, A., 1966. Mutual shading as a factor limiting the yield response of rice to application of nitrogen, phosphorus and potassium fertilizers. Plant Soil 25, 201–210. Tang, Q.Y., 2003. Grain yield construction and N fertilizer efficiency of super hybrid rice under different N applications. Hybrid Rice 18, 44–48. Wang, F., Cheng, F., Zhang, G., 2006. The relationship between grain filling and hormone content as affected by genotype and source-sink relation. Plant Growth Regul. 49, 1–8. Wang, S., Liu, S., Wang, Q., Ding, Y., Huang, P., Ling, Q., 2002a. Relationship between yield formation and leaf nitrogen content and color in rice plant. J. Nanjing Agric. Univ. 25, 1–5. Wang, X., Tao, L., Yu, M., Huang, X., 2002b. Physiological model of super hybrid rice Xieyou 9308. Chin. J. Rice Sci. 16, 38–44. Wang, Z., Gu, Y., Zheng, Y., Wang, H., 2012. Structure observation of rice endosperm cell
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High nitrogen input causes poor grain filling of spikelets at the panicle base of super hybrid rice Penghao Fua, Jing Wanga, Tong Zhanga, Jianliang Huanga,b, Shaobing Penga,
T
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a National Key Laboratory of Crop Genetic Improvement, MARA Key Laboratory of Crop Ecophysiology and Farming System in the Middle Reaches of the Yangtze River, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China b Hubei Collaborative Innovation Center for Grain Industry, Yangtze University, Hubei, 434023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Grain filling High nitrogen input Large-panicle cultivar Rice Spikelet position
It is generally accepted that poor grain filling of rice plants under high nitrogen (N) input is due to increased competition for assimilates because high N rate usually results in large sink size with more spikelets per panicle. In this study, we examined grain filling parameters of spikelets in different panicle position under various N treatments to determine the effect of N rates on grain filling. A super rice cultivar with large panicle was grown under field conditions in Wuxue County, Hubei Province, central China in 2013 and 2014. Fully filled spikelet percentage measured at the crop level from 12 hills decreased by 10.9% when the total N rate increased from 0 to 250 kg ha−1, whereas spikelets per panicle remained almost constant across the N rates. At the individual panicle level, reduction in grain filling was evidenced by decreased fully filled spikelet percentage, fertilized spikelet weight, and grain plumpness, and increased partially filled spikelet percentage in the bottom part of the panicle as total N rates increased. Reduction in these grain filling parameters under high N input was relatively small in the middle part of the panicle and was not appeared in the top part of the panicle. Furthermore, high N rates at both mid-tillering and panicle initiation reduced fertilized spikelet weight in the bottom part of the panicle without an increase in spikelets per panicle. Our results suggest that the spikelets in the bottom part of the panicle were mainly responsible for poor grain filling under high N input and such high N-induced poor grain filling was not due to increased spikelets per panicle.
1. Introduction Rice (Oryza sativa L.) is one of the world’s most important crops and the foremost staple food in Asia (GRiSP, 2013). In China, rice accounts for one-third of the total grain production, which is important for national food security (Xie, 2007). China has successfully bred and released many super rice cultivars to meet the food demands of the rapidly growing population (Cheng et al., 2007). Most of these cultivars have the common characteristics of large panicles (large number of spikelets per panicle), which is one of the most important contributors to high grain yield (Sheehy et al., 2001). However, super rice cultivars do not always meet the expectation in yield performance due to poor grain filling (Huang et al., 2008). The degree of grain filling in rice spikelets can be characterized by the percentage of spikelet filling, the final weight of filled spikelets, and grain plumpness. The degree and rate of grain filling in rice spikelets differ largely with their positions on a panicle (Zhang et al., 2010). Based on their flowering date and position within a panicle, the
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spikelets can be classified as superior and inferior spikelets (Zhu et al., 1988). Superior spikelets flower earlier and are generally located on the top part of the panicle, while inferior spikelets flower later and are generally located on the bottom part of the panicle (Yang et al., 2006). Superior spikelets usually exhibit greater filling rate and heavier final weight than inferior ones (Zhang et al., 2010). Yang and Zhang (2010) reported that the poor grain-filling problem in inferior spikelets is more aggravated in the super rice cultivars. Nitrogen (N) is one of the most important nutrients for the growth and development of rice plants. It can significantly increase grain yield by expanding source and sink capacity (Ding et al., 2014; Fageria and Santos, 2015). However, a higher amount of N than the minimum required to maintain maximum crop growth is often applied to maximize rice grain yield (Lemaire and Gastal, 1997). This problem is more common in China because the average rate of N-fertilizer application for rice in 2006 was 193 kg ha−1, which is approximately 90% higher than the world average (Peng et al., 2010). Over application of N fertilizer is more severe when super rice cultivars are planted because
Corresponding author. E-mail address: [email protected] (S. Peng).
https://doi.org/10.1016/j.fcr.2019.107635 Received 25 July 2019; Received in revised form 22 September 2019; Accepted 23 September 2019 0378-4290/ © 2019 Published by Elsevier B.V.
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lodging does not happen easily with super rice cultivars (Wang et al., 2002b). Peng et al. (2010) reported that there was no positive correlation between grain yield and the amount of N applied based on a large dataset across locations and years in China. Yield reduction is often observed when the high rates of N fertilizer are applied even for the super rice cultivars (Tang, 2003; Liang et al., 2015). A decrease in grain yield under excessive N application was usually attributed to increased susceptibility to lodging (Duy et al., 2004), damage from pests and diseases (Wu et al., 2012), and mutual shading (Tanaka, 1966). Reduction in rice grain filling under high N input was observed in several studies (Ohnishi et al., 1999; Yang et al., 2001b; Jiang et al., 2016), which could be the most direct explanation for the decreased grain yield caused by excessive N application. However, researchers seldom attribute high-N induced yield decline to poor grain filling because high N input can also increase spikelets per panicle (Kamiji et al., 2011; Ding et al., 2014). The degree of grain filling can be reduced under increased sink size due to competition for assimilates (Li et al., 2013). For example, Jiang et al. (2016) reported that spikelet filling decreased by 7 percentage points while spikelets per panicle increased by 25% when N rate at panicle initiation (PI) increased from zero to 180 kg ha−1. In this case, the effect of high N rate on grain filling was confounded by the increased spikelets per panicle. Therefore, the effect of high N input on grain filling should be determined when sink size defined as spikelets per panicle is not increased significantly by high N rates. In this study, the effect of N input on the grain filling of a super rice cultivar was examined when N rates did not affected spikelets per panicle significantly. The objectives of this study were (1) to determine the effect of N rates at different growth stage on the grain filling of a super rice cultivar with large panicles; and (2) to compare spikelets of different panicle position in their grain filling under high N rates.
Table 1 Nitrogen treatments and their application rates (kg ha−1) as basal and topdressing at mid-tillering (MT) and panicle initiation (PI). N treatments
Basal
MT
PI
Total N
0-0-0 0-0-40 0-0-80 90-0-0 90-0-40 90-0-80 90-40-0 90-40-40 90-40-80 90-80-0 90-80-40 90-80-80
0 0 0 90 90 90 90 90 90 90 90 90
0 0 0 0 0 0 40 40 40 80 80 80
0 40 80 0 40 80 0 40 80 0 40 80
0 40 80 90 130 170 130 170 210 170 210 250
Guangzhan63-4S as the female parent and Yangdao 6 as the male parent (Dai et al., 2005). YLY6 was released by the ministry of agriculture as a super hybrid rice cultivar in 2009. The seedlings were raised in a seedbed with a sowing date of May 12 for both years. The soil was plowed and puddled before transplantation. Transplanting was done on June 12, 2013 and June 16, 2014 at a density of 25 hills m−2 and a hill spacing of 13.3 × 30.0 cm with two seedlings per hill. Phosphorus (40 kg P ha-1 as calcium superphosphate) and zinc (5 kg Zn ha-1 as zinc sulfate heptahydrate) were applied at 1 d before transplanting. Potassium (200 kg K ha-1 as potassium chloride) was split equally and applied at 1 d before transplanting and at PI. To minimize leakage between plots, all bunds were covered with plastic film and the plastic film was installed to a depth of 30 cm below soil surface. The paddy fields were flooded after transplanting, and a floodwater depth of 3–5 cm was maintained until 1 week prior to harvest except that the water was drained at maximum tillering stage for about 1 week to reduce unproductive tillers. To prevent biomass and yield losses, pests, diseases, and weeds were intensively controlled throughout the rice growth period in both years.
2. Materials and methods 2.1. Site description Experiments were conducted in farmers’ fields at Lanjie Village, Huaqiao Township, Wuxue County, Hubei Province, China (30°00′ N, 115°44′ E) during the rice-growing season from May to October in 2013 and repeated in an adjacent field in 2014. Soil samples from the upper 20 cm layer were collected for soil analysis before basal fertilizer was applied in both years. The average values of soil properties for the experimental plots across the two years were as follows: a pH of 5.6, organic matter of 32.9 g kg−1, total N of 2.48 g kg−1, Olsen-P of 9.38 mg kg−1, and available K of 112.9 mg kg−1. In both years, climate data were collected during the growing period from transplanting to maturity from a weather station (AWS 800, Campbell Scientific, Inc., USA), located about 100-meter distance from the experimental field. Seasonal average daily minimum temperature, maximum temperature, and solar radiation were 24.6 °C, 32.2 °C, and 17.7 MJ m-2, respectively in 2013 and 23.0 °C, 30.1 °C, and 14.4 MJ m-2, respectively in 2014.
2.3. Sampling and measurement At heading, 100 panicles with similar development stage were labeled on the same day in each plot. Six labeled panicles per plot were sampled at 3-day intervals from heading to maturity. Based on panicle length, the panicles were divided into three equal parts: top, middle, and bottom. Each part was immediately dried in an oven at 80 °C to cease its metabolic activity. Then, all spikelets were detached from panicles by hands. For spikelets sampled at heading, spikelet projection area of 100 spikelets per plot was measured using an Epson Perfection V700 Photo scanner at 300 DPI resolution. The photographs were analyzed using ImageJ 1.6.0 software to determine the length, width, and area of the spikelet. Starting at 3 d after heading, partially filled spikelets were separated from unfilled spikelets by visual observation plus pinching with fingers. For the final samples at maturity, all spikelets without unfilled ones were submerged in a sodium chloride solution with the specific gravity of 1.10 to separate high density grains from others. The floating samples were then submerged in tap water to separate fully filled spikelets from partially filled ones. Spikelet numbers of each category from the top, middle, and bottom parts of the panicle were counted and then samples were dried in an oven at 80 °C to constant weight. In this paper, we divided spikelets into high density grains, fully filled spikelets (including high density grains), partially filled spikelets, and unfilled spikelets. For convenience, all spikelets except for unfilled ones were referred to fertilized spikelets in this study. Grain plumpness was calculated as percent of the dry weight of fertilized spikelets to that of high density grains (Zhu et al., 1995). Because high density grain weights varied across panicle position, the grain plumpness of top,
2.2. Experimental design and crop management The experiments with twelve N treatments were laid out in a completely randomized block design with four replications. Plot size was 35.75 m2 (5.5 × 6.5 m). The twelve N treatments were consisted of various rates and timing of N applied as basal fertilizer (manually broadcast and incorporated in plots 1 d before transplanting) and as topdressing at mid-tillering (MT) and PI. Two N rates with 0 and 90 kg ha−1 were applied as basal and three N rates with 0, 40, and 80 kg ha−1 were applied at MT or PI. The total amount of N application ranged from 0 to 250 kg N ha−1. All N fertilizers were applied in the form of urea. Detailed information about N treatments was listed in Table 1. A widely grown rice cultivar in central China, Yangliangyou 6 (YLY6) with large panicles, was used as the experimental material. YLY6 is an indica hybrid cultivar developed by two-line system in 2001 with 2
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Table 2 Analysis of variance for grain yield and yield components. Source
Grain yield
Panicles m−2
Spikelets per panicle
Spikelets m−2
Fully filled spikelet %
Fully filled spikelet wt
Year N Year × N
* ** ns
** ** **
ns ns ns
ns ** ns
* ** ns
** ** ns
* and **, significant at the 0.05 and 0.01 probability levels, respectively; ns, not significant at 0.05 probability level.
Fig. 1. Responses of grain yield and yield components to total nitrogen rates. Grain yield and yield components were determined at maturity based on the samples of 5 m2 and 12 hills per plot, respectively. Data were averaged across the two years. Error bars indicate ± SE (n = 4).
oven-dried at 80 °C to constant weight. Spikelets per panicle (total spikelets m-2 / panicles m−2) and fully filled spikelet percentage (100 ×fully filled spikelets m−2 / total spikelets m−2) were calculated. Source-sink ratio was calculated as the ratios of dry matter accumulation (DMA) at heading, maturity, and between heading and maturity to the product of total spikelets m−2 and the weight of high density grain. Yield was determined from a 5-m2 area at maturity in each plot and adjusted to the standard moisture content of 0.14 g H2O g-1 fresh weight. Grain moisture content was measured with a digital moisture tester (DMC-700, Seedburo, Chicago, IL, USA). Plant N concentration of the samples for yield component was determined by an elemental analyzer (Elementar vario MAX CNS/CN, Elementar Trading Co., Ltd, Germany). Total N uptake was the summation of N content of straw, fully filled spikelets, and the combined sample of rachis, partially filled spikelets, and unfilled spikelets.
middle, and bottom parts of the panicle was calculated based on the high density grain weights of the three parts of the panicle, respectively. Twelve hills were sampled from each plot at maturity to measure yield components. Plant samples were separated into straw and panicle after recording panicle numbers. Dry weight of straw was determined after oven-dried at 80 °C to constant weight. Panicles were hand-threshed and fully filled spikelets were separated from others by submerging them in tap water. Unfilled spikelets were separated from partially filled ones by winnowing. Three subsamples with each of 30 g fully filled spikelets and 2 g unfilled spikelets were taken to determine the numbers of fully filled and unfilled spikelets, whereas the entire sample was counted to determine the number of partially filled spikelets. The numbers of fully filled, partially filled, and unfilled spikelets were added to determine total spikelets m−2. Dry weights of rachis, fully filled, partially filled, and unfilled spikelets were determined after 3
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Fig. 2. Responses of fertilized, fully filled, and partially filled spikelet percentages of the top (T), middle (M), and bottom (B) parts of the panicle to total nitrogen rates in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
did not result in an increase in grain yield (Fig. 1a, Table S1). The yield response to the N rate was mainly explained by the difference in spikelets m−2 across N treatments (Fig. 1b), which was determined primarily by panicles m−2 (Fig. 1c), not by spikelets per panicle (Fig. 1d). In fact, spikelets per panicle remained almost constant across the N rates. In contrast, there was a declining trend in fully filled spikelet percentage with the increase in total N rate (Fig. 1e). Fully filled spikelet percentage decreased by 10.9% when the total N rate increased from 0 to 250 kg ha-1 (Table S1). A slight increase in fully filled spikelet weight was observed when the total N rate increased from 0 to 90 kg ha1 , and further increase of the N rate did not affect this trait significantly (Fig. 1f). The responses of DMA, tissue N concentration, and total N uptake were similar between 2013 and 2014 since the interactive effects between year and N treatments were insignificant for these traits (Table S2). Dry matter accumulation (DMA) at heading and maturity increased with the increase in total N rate, whereas DMA between heading and maturity remained relatively stable across the N treatments (Fig. S1a-c, Table S3). Nitrogen concentrations of straw and fully filled spikelets increased with the increase in total N rate (Fig. S3a-b, Table S3). This was also true for total N uptake (Fig. S3c, Table S3). Fertilized spikelet percentage remained fairly constant across total
2.4. Statistical analysis Statistical data analysis was performed using analysis of variance (Statistix 9.0, Analytical Software, Tallahassee, FL, USA). Year, N management, and panicle position were considered fixed effects and block was considered a random effect. When significant, the mean values were compared by the least significance difference (LSD) test at the 0.05 probability level. For variables without significant year × N treatment interactions, data were presented with means across the two years. The responses of agronomic and grain filling traits to total N rates were fitted with linear or quadratic functions, while the time-course of fertilized spikelet weight was fitted with Richards’ growth equation (Richards, 1959). 3. Results Grain yield and yield components except for spikelets per panicle were significantly affected by the N treatments, whereas the interactive effects between year and N treatments were insignificant for grain yield and yield components except for panicles m−2 (Table 2). Average grain yield across the two years increased with the increase in total N rate until the N rate reached 210 kg ha-1 and further increase of the N rate 4
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Fig. 3. Responses of fully filled and fertilized spikelet weights of the top (T), middle (M), and bottom (B) parts of the panicle to total nitrogen rates in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
rates and years, fertilized spikelet weight of the bottom part of the panicle was 17.5% lower than that of the top part of the panicle. Figs. 4 and 5 showed the time-course of grain development from heading to maturity for the three N rates of 0, 40, and 80 kg ha−1 applied at MT and PI, respectively. In the top part of the panicle, zero N rate at MT had higher fertilized spikelet weight than the N rates of 40 and 80 kg ha−1 at MT during the early phase of grain development, and the difference disappeared when fertilized spikelet weight reached maximum level (Fig. 4a, d). Similar response of fertilized spikelet weight to the N rate at MT was observed in the middle part of the panicle except for the late phase of grain development in 2013 when the N rates of 0 and 40 kg ha−1 at MT had higher fertilized spikelet weight than the N rate of 80 kg ha−1 at MT (Fig. 4b, e). In the bottom part of the panicle, the three N rates had similar fertilized spikelet weight up to 12 d after heading, and since then the high N rate at MT reduced fertilized spikelet weight significantly, especially in the late phase of grain development (Fig. 4c, f). The N rates at PI had smaller effect on grain filling during the early phase of grain development than that at MT in the top and middle parts of the panicle (Figs. 4 and 5). Fertilized spikelet weight was not affected by the N rate at PI in the top part of the panicle in both years and in the middle part of the panicle in 2014 (Fig. 5a, d, e). The N application at PI reduced fertilized spikelet weight significantly in the late phase of grain development in the middle part of the panicle in 2013 (Fig. 5b). In the bottom part of the panicle, the N rates at PI had a similar effect on fertilized spikelet weight as the N rates at MT (Fig. 5c, f). Therefore, excessive N application at MT and PI had significantly negative effect on the grain filling of the bottom part of the panicle. Grain plumpness decreased with the increase in total N rate in the bottom and middle parts of the panicle, whereas the grain plumpness of the top part of the panicle remained relatively constant across total N rates (Fig. 6a-b). The grain plumpness of the top part of the panicle was maintained at more than 94% regardless of the N rates, whereas grain plumpness of the middle and bottom parts of the panicle reduced to as low as 85.7% and 64.4%, respectively, when the total N rate increased from 0 to 250 kg ha−1. Difference between the top and middle parts of
N rates, especially in 2013 (Figs. 2a, d), this was evidenced by the insignificant effect of the N treatment on this trait in 2013 (Table S4). The bottom part of the panicle had lower fertilized spikelet percentage than the top and middle parts of the panicle. Such difference was more consistent in 2013 than 2014. There was no difference in fertilized spikelet percentage between the top and middle parts of the panicle in 2013, and the difference was significant only at the N rate of 250 kg ha−1 in 2014. Fully filled spikelet percentage decreased while partially filled spikelet percentage increased with the increase in total N rate except for the top part of the panicle in 2013 (Fig. 2b-c, e-f). This was supported by the fact that the interactive effects between the N treatment and panicle position were significant for both fully and partially filled spikelet percentages (Table S4). In general, the bottom part of the panicle showed the greatest responses to high N input in fully and partially filled spikelet percentage, followed by the middle and top parts of the panicle. The bottom part of the panicle had lower fully filled spikelet percentage and higher partially filled spikelet percentage than the top and middle parts of the panicle across N treatments. There was no difference in fully filled spikelet percentage and partially filled spikelet percentage between the top and middle parts of the panicle under low N conditions, but the difference was significant under high N conditions. Averaged across the N rates and years, fully filled spikelet percentage of the top part of the panicle was 56.5% higher than that of the bottom part of the panicle, while partially filled spikelet percentage in the top part of the panicle was only 20.9% of that of the bottom part of the panicle. Fully filled spikelet weight showed a slightly increasing trend with the increase in total N rate and the top part of the panicle had the highest fully filled spikelet weight, followed by the middle and bottom parts of the panicle (Fig. 3a, c). Fertilized spikelet weight decreased with the increase in total N rate in the middle and bottom parts of the panicle in 2013 and only in the bottom part of the panicle in 2014 (Fig. 3b, d). The bottom part of the panicle had lower fertilized spikelet weight than the top and middle parts of the panicle. Difference between the top and middle parts of the panicle in fertilized spikelet weight was significant only at the N rate of 250 kg ha−1. Averaged across the N 5
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Fig. 4. Changes in fertilized spikelet weights of the top (T), middle (M), and bottom (B) parts of the panicle during grain filling period for the nitrogen rates of 0, 40, and 80 kg N ha−1 applied at mid-tillering in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
maturity did not decrease across the N rates (Fig. S1c), and (3) reduction in source-sink ratios calculated at heading, maturity, and between heading and maturity was not observed consistently across the two years (Fig. S2). Furthermore, the poor grain filling under high N conditions was not due to the N deficiency of rice crop because high N concentrations of straw and fully filled spikelets and high total N uptake were observed under high N input (Fig. S3). At the individual panicle level, reduction in grain filling was evidenced by decreased fully filled spikelet percentage, fertilized spikelet weight, and grain plumpness, and increased partially filled spikelet percentage in the bottom part of the panicle as total N rate increased (Figs. 2,3, and 6). Reduction in these grain filling parameters under high N input was relatively small in the middle part of the panicle and was not appeared in the top part of the panicle. The reduction in fully filled spikelet percentage and grain plumpness in the bottom part of the panicle was unlikely due to increased spikelet size under high N input because spikelet area remained stable across the N rates for all three parts of the panicle (Fig. S4). Furthermore, fertilized spikelet weight during grain-filling period was compared among the three N rates (0, 40, and 80 kg ha−1) at MT and PI. High N rates at both MT and PI reduced fertilized spikelet weight in the bottom part of the panicle (Figs. 4 and 5). Again, high N rates at both MT and PI did not increase
the panicle in grain plumpness was significant only at the N rate of 250 kg ha−1. 4. Discussion Reduction in grain filling under high N input was observed in this study at both crop and individual panicle levels. More importantly, the reduced grain filling was not due to increased sink size because spikelets per panicle remained relatively stable across the N rates. At the crop level, fully filled spikelet percentage decreased by 10.9% when the total N rate increased from 0 to 250 kg ha−1, while spikelets per panicle did not increase across the N rates based on the data from 12-hill samples (Fig. 1). Fully filled spikelet weight did not decrease significantly when the total N rate increased from 130 to 250 kg ha−1. These results suggest that the stagnation of rice grain yield under high N conditions was mainly caused by poor grain filling. This result is in consistent with Jiang et al. (2016) who reported reduction in grain filling under the highest N rate of 180 kg ha-1 applied at PI. The poor grain filling under high N conditions was unlikely due to increased competition for assimilates because of the following reasons: (1) dry matter accumulation (DMA) at heading and maturity increased with the increase in total N rate (Fig. S1a-b), (2) DMA between heading and 6
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Fig. 5. Changes in fertilized spikelet weights of the top (T), middle (M), and bottom (B) parts of the panicle during grain filling period for nitrogen rates of 0, 40, and 80 kg N ha−1 applied at panicle initiation in 2013 and 2014. Fertilized spikelets include all spikelets except for unfilled ones. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
Fig. 6. Responses of grain plumpness of the top (T), middle (M), and bottom (B) parts of the panicle to total nitrogen rates in 2013 and 2014. Data were from 6 panicles per plot sampled at maturity. Error bars indicate ± SE (n = 4).
rice plants (Yao et al., 2000; Fukushima et al., 2011; Jiang et al., 2016). High N application at vegetative stage increases spikelets m−2 by promoting tillering and panicles m−2 (Sui et al., 2013), while high N application at PI increases sink size by enhancing spikelets per panicle
spikelets per panicle (Table S5), suggesting that the reduced fertilized spikelet weight in the bottom part of the panicle under high N input was not due to increased sink size. Increased sink size under high N input was commonly observed in 7
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(Ding et al., 2014). In our study, spikelets m−2 reached almost maximum level when total N rate increased to 130 kg ha-1 and spikelets per panicle remained stable across all N rates. The lack of response of sink size to high N input might be due to high indigenous N supply of the soil in our study which was evidenced by the fact that over 7 t ha-1 of grain yield was produced without N application. Wang et al. (2002a) reported that the spikelets per panicle of tiller stems began to decrease sharply when the N concentration in the top two leaves increased to a certain level, which might explain the lack of response of sink size to increased N rates. High N input also resulted in high N accumulation and enhanced N metabolism in rice plant and thereby delayed and decreased non-structural carbohydrate accumulation, which could reduce spikelets per panicle (Li et al., 2010). Our study indicates that the spikelets in the bottom part of the panicle were mainly responsible for reduced grain filling under high N input. Poor grain filling of spikelets in the bottom part of the panicle has been reported for cultivars with large panicles such as intersubspecific hybrid between indica and japonica (Liang et al., 2001). These spikelets are considered as inferior ones because they usually exhibit lower filling rate and lighter final weight than superior ones (Yang et al., 2006). Over the last few decades, considerable effort has been made to elucidate the mechanisms underlying poor grain filling of spikelets in the bottom part of the panicle and to identify a solution for this problem. It has been suspected that the poor grain filling of inferior grains may be due to the lack of assimilate supply (Li et al., 2013), weak sink strength (Liang et al., 2001), endogenous hormone inhibition (Wang et al., 2006), low activity of key enzymes associated with sucrose–starch metabolism (Yang et al., 2001a), and poor transport capacity (Huang et al., 2005). The precise mechanism and strategies that could be used to enhance grain filling remain unclear (Yang and Zhang, 2010). In the present study, we demonstrated that poor grain filling of the bottom part of the panicle became a limiting factor for further increase in rice grain yield under high N treatments. As stated above, poor grain filling of the basal part of the panicle under high N application rates was not attributed to reduction in source-sink ratio, N deficiency, increased sink size, or increased spikelet size. Previous studies reported that ethylene had an adverse effect on the grain filling of rice, especially for cultivars with large panicles because of the longer period of exposure of the lower part of the large panicles to the gaseous ethylene (Mohapatra and Panigrahi, 2011; Yang and Zhang, 2005). Ethylene inhibited the partitioning of assimilate in developing spikelets and resulted in low starch biosynthesis and high accumulation of soluble carbohydrate (Sekhar et al., 2015). The spikelets in the bottom part of the panicle initiated earlier but flowered later than the spikelets in the top part of the panicle, which meant longer exposure in ethylene environment for the spikelets in the bottom part of the panicle (Ikeda et al., 2004). High N input might aggravate the ethylene effect on spikelets in the bottom part of the panicle through prolonging the ethylene exposure duration, because high N input always cause longer panicle development duration (Liu et al., 2005). Yang and Zhang (2005) stated that when plant senescence is unfavorably delayed under high N conditions, rice will exhibit prolonged and slow grain filling. High N application was accompanied by an increased tiller number and leaf area, which led to mutual shading, canopy closure and reduced light penetration into the rice canopy (Tanaka, 1966). Excessive N concentrations in plant tissue might result in a high rate of N metabolism and overconsumption of carbohydrate, thereby reducing carbohydrate supply for grain filling (Li et al., 2010; Liang et al., 2015). Given that carbohydrate is the main composition of endosperm, the carbohydrate acceptance capacity of the endosperm may also play a vital role in grain filling (Wang et al., 2012). The cell number, size, and filling degree of endosperm are all closely related to grain weight and grain filling (Yang et al., 2006; Li et al., 2013). Nevertheless, the physiological mechanism underlying poor grain filling of the bottom part of the panicle under high N input should be further studied in future.
5. Conclusions This study showed that high N input induced poor grain filling of spikelets at the panicle base. Fully filled spikelet percentage, fertilized spikelet weight, and grain plumpness decreased, while partially filled spikelet percentage increased in the bottom part of the panicle as total N rates increased. The high N-induced poor grain filling observed in this study was not due to increased sink size because spikelets per panicle remained relatively stable across the N rates. Our results suggest that high N input could reduce the grain filling of rice crop without the increased spikelets per panicle. Declaration of Competing Interest There are no conflicts of interest to declare. Acknowledgements This work was supported by the National Key Research and Development Program of China (2016YFD0300210) and a grant from the National Natural Science Foundation of China (No. 31671620). We acknowledge the assistance from staff members of the Modern Agricultural Demonstration Center of Wuxue County. References Cheng, S., Cao, L., Zhuang, J., Chen, S., Zhan, X., Fan, Y., Zhu, D., Min, S., 2007. Super hybrid rice breeding in China: achievements and prospects. J. Integr. Plant Biol. 49, 805–810. Dai, Z., Liu, G., Li, A., Xu, M., Liu, X., Zhou, C., Zhang, H., 2005. Breeding of two-line indica hybrid rice combination, "Yangliangyou 6″, and studying on its culture characteristics. Chin. Agric. Sci. Bull. 21, 114–116. Ding, C., You, J., Chen, L., Wang, S., Ding, Y., 2014. Nitrogen fertilizer increases spikelet number per panicle by enhancing cytokinin synthesis in rice. Plant Cell Rep. 33, 363–371. Duy, P.Q., Abe, A., Hirano, M., Sagawa, S., Kuroda, E., 2004. Analysis of lodging-resistant characteristics of different rice genotypes grown under the standard and nitrogen-free basal dressing accompanied with sparse planting density practices. Plant Prod. Sci. 7, 243–251. Fageria, N.K., Santos, A.B., 2015. Yield and yield components of lowland rice genotypes as influenced by nitrogen fertilization. Commun. Soil Sci. Plant Anal. 46, 1723–1735. Fukushima, A., Shiratsuchi, H., Yamaguchi, H., Fukuda, A., 2011. Effects of nitrogen application and planting density on morphological traits, dry matter production and yield of large grain type rice variety bekoaoba and strategies for super high-yielding rice in the Tohoku region of Japan. Plant Prod. Sci. 14, 56–63. GRiSP (Global Rice Science Partnership), 2013. Rice Almanac, 4th edition. International Rice Research Institute, Los Baños, the Philippines 283 p. Huang, M., Mo, R., Zou, Y., Mo, Y., Zhan, K., Jiang, P., 2008. Yield components and grain filling characters of super hybrid rice. Crop Res. 22, 249–253. Huang, S., Zou, Y., Liu, C., 2005. Setting physiology of the superior and inferior grains of hybrid rice Liangyoupeijiu. Acta Agron. Sin. 1, 102–107. Ikeda, K., Sunohara, H., Nagato, Y., 2004. Developmental course of inflorescence and spikelet in rice. Breed. Sci. 54, 147–156. Jiang, Q., Du, Y., Tian, X., Wang, Q., Xiong, R., Xu, G., Yan, C., Ding, Y., 2016. Effect of panicle nitrogen on grain filling characteristics of high-yielding rice cultivars. Eur. J. Agron. 74, 185–192. Kamiji, Y., Yoshida, H., Palta, J.A., Sakuratani, T., Shiraiwa, T., 2011. N applications that increase plant N during panicle development are highly effective in increasing spikelet number in rice. Field Crops Res. 122, 242–247. Lemaire, G., Gastal, F., 1997. N uptake and distribution in plant canopies. In: Lemaire, G. (Ed.), Diagnosis of the Nitrogen Status in Crops. Springer, Berlin Heidelberg, Berlin, Heidelberg, pp. 3–43. Li, G., Wang, H., Wang, S., Wang, Q., Zheng, Y., Ding, Y., 2010. Effect of nitrogen applied at rice panicle initiation stage on carbon and nitrogen metabolism and spikelets per panicle. J. Nanjing Agric. Univ. 1, 1–5. Li, X., Cheng, H., Wang, N., Yu, C., Qu, L., Cao, P., Hu, N., Liu, T., Lyu, W., 2013. Critical factors for grain filling of erect panicle type japonica rice cultivars. Agron. J. 105, 1404–1410. Liang, J., Zhang, J., Cao, X., 2001. Grain sink strength may be related to the poor grain filling of indica-japonica rice (Oryza sativa L.) hybrids. Physiol. Planta. 112, 470–477. Liang, Z., Bao, A., Li, H., Cai, H., 2015. The effect of nitrogen level on rice growth, carbonnitrogen metabolism and gene expression. Biologia 70, 1340–1350. Liu, X.W., Meng, Y.L., Zhou, Z.G., Cao, W.X., 2005. Dynamic characteristics of floret differentiation and degeneration in rice. Acta Agron. Sin. 314, 451–455. Mohapatra, P., Panigrahi, R., 2011. Ethylene control of grain development in the inferior spikelets of rice panicle. Adv. Plant Physiol. 12, 79–89. Ohnishi, M., Horie, T., Homma, K., Supapoj, N., Takano, H., Yamamoto, S., 1999. Nitrogen management and cultivar effects on rice yield and nitrogen use efficiency in
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