TIAN Qing-lan ,HE Lian-huaLIAO ShuangLI WuDENG FeiZHOU WeiZHONG Xiao-yuanREN Wan-jun

1 Key Laboratory of Crop Ecophysiology and Farming System in Southwest China/Crop Ecophysiology and Cultivation Key Laboratory of Sichuan Province/College of Agronomy,Sichuan Agricultural University,Chengdu 611130,P.R.China

2 Biotechnology Research Institute,Guangxi Academy of Agricultural Sciences,Nanning 530007,P.R.China

Abstract The yield and nitrogen use efficiency (NUE) of hybrid rice combinations are closely related to restorer line. Therefore,it is essential to evaluate the agronomic characteristics of restorer lines with high yield and high NUE (HYHN). However,it is unclear which restorer lines are HYHN,and neither have the common agronomic traits of the HYHN restorer lines been identified. Aiming to address this issue,we conducted two filed experiments using three nitrogen applications,which screened five HYHN restorer lines from 15 indica restorer lines. Yield,NUE and nutrient transportation of restorer lines with different yields and NUE types were examined. Yield and total nitrogen absorption in aboveground biomass (TNA)increased,whereas NUE for grain production decreased with increasing nitrogen application levels. The HYHN restorer lines had large spikelets and high weight per panicle that were significantly positively correlated with yield and NUE. Therefore,large sink potential may be beneficial for both yield and NUE. We further studied the differences in nutrient transportation to panicles between the HYHN and low yield and low NUE (LYLN) restorer lines and found that the former had a higher nitrogen absorption level and dry matter weight ratios of panicle in maturity. Moreover,the HYHN lines also had a higher root and neck-panicle node bleeding intensity per stem after heading and more developed vascular bundles of neck-panicle nodes and leaves than the LYLN lines,which could contribute to the transportation of nutrients from root to ground and from stem and leaf to spike. Therefore,the advantages of large sink potential of the HYHN restorer lines include large nutrient accumulation in and distribution to the panicles and smooth flow of nutrients along the transportation channels.

Keywords:indica hybrid rice,restorer lines,grain yield,nitrogen-use efficiency,sink potential,nutrient transportation

1.Introduction

The projected demands for increased crop yields due to increasing population,shifting dietary preferences and high biofuel consumption require global crop production to be doubled by 2050 (Rayet al.2013). Rice is a staple crop for approximately half of the world’s population (Godfrayet al.2010) and plays an important role in global food security.China is one of the largest rice producers worldwide,accounting for 18.5 and 27.7% of the global planted area and production,respectively (Zhuet al.2015).

Nitrogen is one of the most important elements for plant growth,and the application of nitrogen fertilizers plays important roles in increasing rice yield (Zhanget al.2003;Panet al.2012;Conget al.2015). However,in China,over application of nitrogen fertilizer has resulted in wasted resources (Penget al.2006),soil acidification (Guoet al.2010) and water pollution (Barton and Colmer 2006),among other issues. In particular,high nitrogen fertilizer application has reduced the nitrogen recovery efficiency of rice in China,which is lower than the world average (46%) and considerably lower than that reported by several European countries (70%) (Ladhaet al.2005;Liuet al.2010). The average pure nitrogen application in China is 180 kg ha-1,which is approximately 75% higher than the world average(Penget al.2006). These results highlight low nitrogen-use efficiency (NUE) in China. As a response,since 2015,the Ministry of Agriculture of China has been promoting“Action Plan for Zero-growth of Fertilizer Use by 2020”is necessary to improve NUE to ensure higher rice production in China.

Cultivation measures,such as selection of fertilizer type,balanced fertilization (Chenet al.2015;Denget al.2016;Liet al.2017;Xiaet al.2017),fertilization methods,and fertilizer management (Yaoet al.2012;Liuet al.2013),can improve NUE of rice to some extent and ensure a steady grain yield. However,the potential yields and NUEs differ among rice varieties. It therefore becomes essential to breed varieties with high yield and high NUE(HYHN). Genetic and molecular approaches can be used to improve NUE of rice (Kantet al.2010;Weiet al.2012;Daiet al.2015),as well as to increase its yield (Yuet al.2012). However,studies on the genetic improvement of NUE in rice have mainly focused on screening genotypes with high NUE and its coming ability of dry matter production and other aspects (Xuet al.2009). Although different rice varieties can be screened and popularized (Fenget al.2014;Yuet al.2015),there are no standardized methods for the classification of HYHN varieties owing to the ambiguous definition of high NUE.

The differences in NUE are universal and broad,between conventional rice and hybrid rice,betweenindicaandjaponicasubspecies,and even among different varieties(lines) within the same subspecies (Zhanget al.1989;Wu and Tao 1995). The vast difference in NUE among rice genotypes provides the genetic basis for NUE improvement.The general combing ability (GCA) effect of parents was controlled by the additive effect of genes,which could be stably inherited to the offspring combination (Piaoet al.2005). In a previous study,the parents’ GCA effect could be predicted by the apparent values such as NUE for grain production (NUEg) at different nitrogen levels (Piaoet al.2005). Furthermore,the male parent was shown to have a greater influence on NH4+absorption in the F1generation(Ni and An 1984). The nitrate reductase activity of the F1generation seedlings of hybrid rice is between those of their parents (Linet al.1986). Therefore,as the male parent of three-line hybrid,restorer line had great influence on NUE of F1generation. Xuet al.(2009) regarded that exploring the differences in NUE among representative varieties,hybrid combinations,male sterile lines,and restorer lines is vital for developing rice genotypes with high NUEs. However,the yield and NUE ofindicahybrid restorer lines,particularly under different nitrogen application treatments,have not yet been investigated.

The mechanisms of nitrogen absorption (NA) and translocation are complex and involve nitrogen cycling(particularlyviaamino acids),including the processes from the stem to root through phloem and from the root to stem through xylem (Marsehneret al.1997). The absorption and utilization of nitrogen in plants are known to be interdependent processes (Vinod and Heuer 2012).NO3–absorbed by plants is immediately reduced to NO2–by nitrate reductase by using nicotinamide adenine dinucleotide hydrogen donor,and then reduced to NH4+by nitrite reductase;the reduced NH4+and the NH4+absorbed by plants are required to synthesize amino acidsviathe glutamine synthetase (GS) and glutamate synthase(GOGAT) pathways (Campbell 2002). Hakeemet al.(2011)found that nitrogen-efficient rice cultivars can reduce nitrate pollution. However,nutrient transportation in HYHNindicarestorer lines remains unclear.

Therefore,in this study,we aimed to determine the NUE ofindicarestorer lines exposed to different nitrogen levels in order to select the most efficient HYHN-type restorer lines and to determine their sink potential and nutrient transportation. Our findings provide fundamental information for cultivating hybrid HYHN rice varieties.

2.Materials and methods

2.1.Study site

Two field experiments were conducted in the same field in Sandaoyan Town (30°52´N,103°55´E),Chengdu Plain,Sichuan,China,in 2015 and 2016,respectively. The previous crop in both years was radish. Daily maximum temperature,daily average temperature and in rice growing season rainfall of Sandaoyan Town are shown in Appendix A. Soil was a medium loam with 25.46 g kg–1organic matter,2.46 g kg–1total nitrogen,0.49 g kg–1total phosphorus,7.60 g kg–1total potassium,122.54 mg kg–1alkali-hydrolyzable nitrogen,63.50 mg kg–1available phosphorus,and 115.41 mg kg–1available potassium in the 0–20 cm soil layer.

2.2.Experimental design and field measurements

Nitrogen application level (main plot factor) and rice variety(split plot factor) were investigated using a two-factor splitplot design with three replicates,with 144 and 48 plots in 2015 and 2016,respectively. The main plot factor included no nitrogen application (N0),nitrogen application of 90 kg ha–1(N90) and nitrogen application of 180 kg ha–1(N180).In the first study year (2015),the split plot factor included 15indicahybrid rice restorer lines and anindicahybrid variety(Yixiangyou 2115).In the second study year (2016),the split factor indluded two HYHN restorer lines (Lehui 188 and Shuhui 498) and two low yield and low NUE (LYLN)restorer lines (Yahui 2115 and Duohui 1) selected from 15indicahybrid rice restorer lines in 2016. Detailed information concerning the tested materials is shown in Table 1.

Seeds were sowed on a dry land bed on April 2 in both years.Seedlings were individually transplanted with a spacing of 0.30 m×0.16 m,on May 10,2015 at 38-day-old and May 13,2016 at 41-day-old,respectively. Plot dimensions were 0.9 m×6 m and 3.6 m×6 m in 2015 and 2016,respectively. Growth duration ranged from 156 to 166 d in 2015 and from 154 to 161 d in 2016 (Appendix B).

The field ridge of the main plots was covered with a plastic film to ensure independent management of their water and fertilizer. Due to different growth processes of the tested varieties,except under the N0 treatment,nitrogen fertilizer(urea) was applied at the ratio of 7:3 at the basal and 10 days after transplanting to ensure the consistency of fertilization management of the tested varieties. For each treatment,90 kg ha–1of P2O5as single superphosphate was applied at the basal stage,and 180 kg ha–1of K2O as potassium chloride was split equally and applied at the basal and 10 days after transplanting. Pesticides were used to control insects,pathogens and weeds. Damp irrigation was applied before the panicle initiation stage,rational irrigation from panicle initiation to heading and alternate wetting and drying irrigation after the heading stage. Irrigation was stopped 7 days before harvest.

Table 1 Tested major restorer lines of indica hybrid rice in 2015 and 2016 at Sandaoyan Town,Sichuan,China

2.3.Sampling and measurement

Nitrogen concentrationAt the heading and maturity stages,the tiller number of 30 hills in each plot was counted,and then three representative hills with the mean tiller number were sampled. Sampled plants were divided into leaf lamina,stem plus sheath,and panicle;dried at 105°C for 1 h to inactivate enzymes;and oven-dried at 80°C to obtain a constant mass. Dried samples were weighed,milled and sifted through a 0.5-mm screen. Next,their nitrogen concentrations were determined by FOSS 8400 Automatic Kjeldahl nitrogen determinator (Denmark) (Denget al.2012).

Physiological characteristicsAccording to Zhao (1999)and Liuet al.(2015),two plants from each hill were selected according to the average number of tillers to determine the neck-panicle node and root bleeding intensities at the fullheading stage and at 10,20 and 30 d thereafter,respectively.Panicles were crosscut at the neck-panicle at 6:00 p.m.,and the incision was covered with a weighed cotton ball (about 1 g);cotton balls were removed and weighed at 9:00 a.m.the following day after 15 h. Next,the neck-panicle node bleeding intensity per stem was calculated as the difference of the two values before and after 15 h. The root bleeding intensity was determined in the similar manner except that the crosscut was made at 15 cm from the base.

Vascular bundle of neck-panicle nodes and leaf midribsThe main stems of 10 plants per plot were marked at the tillering stage,six of which were sampled at the fullheading stage. The main stems were cut 1.5 cm below the neck-panicle nodes and fixed with formalin-aceto-alcohol fixative (90 mL of 70% ethanol,5 mL glacial acetic acid and 5 mL of 37% formalin) for 24 h,rinsed with 70% ethanol until all acetic acid was removed,and then desilicated with 10%hydrofluoric acid solution for 15 d. After sample embedding,slicing and safranine fast green staining and production by Wuhan Servicebio Technology Co.,Ltd.,China,paraffin sections were observed under a LEICA-M165 microscope.Top leaves were sampled at the full-heading stage and prepared and observed in the same way. We used Software Kit LAS V4.2 to determine the number and area of macrovascular bundles and small vascular bundles,the area of phloem and xylem of the macro-vascular bundles and small vascular bundles,and the area of cross section and pith cavity. Area of vascular bundles was the sum of the area of macro-vascular bundles and small vascular bundles. Area of culm wall (mm2)=Area of cross section–Area of pith cavity;Ratio of vascular bundle area (%)=Area of vascular bundle/Area of culm wall×100.

Grain yield and its componentsPlots were hand harvested to estimate grain yield on 15 September and 10 September in 2015 and 2016,respectively. According to Tian (2016),about 1 kg wet grain was sampled each year for determining moisture content and grain proportion after removing impurities during the process of air drying.We then determined the actual grain yield of each plot through calculating the residential area,harvested grain yield,moisture content,discount rate,and loss. At maturity,panicle number of 60 hills in each plot was counted to calculate the panicles per unit area,which sampled 5–8 hills having more than 50 panicles were sampled. After they were air-dried,the panicle lengths,empty grain number,total grain weight,and 1 000-grain weight were measured in the laboratory. Then spikelets per panicle (SPP),weight per panicle (WPP),and grain setting rate were calculated:Total grain number=Total grain weight/1 000-grain weight×1 000;Grain setting rate (%)=(Total grain number–Empty grain number)/Total grain number×100.

2.4.Variety screening

At present,the classification standards of high NUE and low NUE varieties were not unified. For example,previous studies differentiated between the high NUE and low NUE varieties according to NUEg (Piaoet al.2004),yield under low nitrogen and high nitrogen conditions (Huanget al.2015;Yuet al.2015),or yield with its components and the leaf SPAD value and NA (Fenget al.2014) to classify high NUE and low NUE varieties. However,single judgment criterion is inadequate to assess the NUE of a variety. In this study,a large amount of data were collected as the basis for variety classification. We selected factor analysis to classify the NUE of varieties,because it can reduce the indexes of different measures to the same dimension,and simplify the complex data for subsequent analysis. Overall,144 plots of grain yield data from 48 treatments applied in 2015 were analyzed in SPSS 18.0 using factor analysis,and a comprehensive score of grain yield was calculated. Based on these treatments,eight NUE indices were calculated:total NA in aboveground biomass (TNA),NA per 100 kg grain(NAG),NUE in biomass production (NUEb),NUEg,nitrogen harvest index (NHI),nitrogen partial factor productivity(NPFP),apparent nitrogen recovery efficiency (ANRE),and physiological NUE (PNUE). Data from these indices were also analyzed using factor analysis. Eleven common factors were selected to calculate the comprehensive score of NUE according to their weights. Respective factor scores of 11 common factors were obtained,and their rotation variance rates and cumulative variance rate (96.065%) after rotation were obtained from the rotation sums of squared loadings.The weights of 11 common factors were calculated to divide the rotation variance rate by the cumulative variance rate.Finally,the comprehensive score of NUE for each variety was obtained by multiplying the factor scores and weights of each common factor. Comprehensive scores greater than zero represented high yield or high NUE,and scores lower than zero represented low yield or low NUE. Finally,based on the comprehensive scores of grain yield and NUE,the tested varieties were divided into four types:HYHN,high yield and low NUE (HYLN),low yield and high NUE (LYHN),and low yield and low NUE (LYLN).

2.5.Data analyses

Data were processed and mapped using Excel.Statistical data were analyzed using analysis of variance. The least significant difference test at the 5% level was used to determine differences between the means of the treatments.Factor analysis and regression analysis were performed using SPSS 18.0 (SPSS Inc.,Chicago,IL,USA).

2.6.Calculations

According to Ye (2005),Peng (1996) and Wu (2016),the NA of organs,TNA,NAG,NUEb,NUEg,NHI,NPFP,ANRE,PNUE,and agronomic NUE (ANUE) were calculated as follows:

3.Results

3.1.Grain yield and NUE of restorer lines

Grain yield ranged from 6.20 to 11.65 t ha-1in 2015 and from 7.03 to 11.61 t ha-1in 2016 (Table 2). N180 produced the highest average grain yield,followed by N90 and N0 in both years. Grain yields under N180 and N90 was 23.10 and 18.67% higher than that under N0,respectively,in 2015.In this year,grain yield was the highest for Yixiangyou 2115(hybrid rice),Lehui 188 and Mianhui 725 under the three nitrogen treatments. Lehui 188 and Shuhui 498 showed significantly higher grain yields than Yahui 2115 and Duohui 1 under the three treatments in both years. In addition,different restorer lines exhibited different responses to nitrogen applications;most restorer lines showed a wider range of increased yield under N0–N90 (8.25–34.62%) than under N90–N180 (–6.51–24.35%),whereas the yields of Luhui 602,Mianhui 523 and Shuhu 527 showed negative trends under N90–N180.

A comparison of NUEs among the three nitrogen application treatments (Table 3) revealed that TNA and NAGunder N180 were higher than those under N90 and N0 in both years. However,NUEb,NUEg and NHI showed an opposite trend in both years,and NPFP and PNUE were significantly higher under N90 than under N180 in both years. Results from the two years showed that NUEg was 56.98–74.79,52.49–69.87 and 43.38–61.20 kg kg-1under N0,N90 and N180,respectively,whereas ANRE was 13.82–55.40% and 16.54–49.53% under N90 and N180,respectively. Moreover,in 2015,TNA,NUEg,NHI,and NPFP of the hybrid rice Yixiangyou 2115 were higher than those of most restorer lines under the three nitrogen application treatments. Among all the restorer lines,Lehui 188 had the highest NUEb,NUEg,NHI,NPFP,PNUE,and ANUE;Luhui 602 had the highest TNA;and Shuhui 498 had the highest NPFP and ANRE,under the three nitrogen application treatments. In 2015 and 2016,the average values of TNA,NUEg,NHI,PNUE,and NPFP of Shuhui 498 and Lehui 188 under the three nitrogen application treatments were higher than those of Yahui 2115 and Duohui 1 (Table 3). Furthermore,ANUE and PNUE of Lehui 188 and Chenghui 727 increased with increasing nitrogen application levels,whereas those of Mianhui 523,Fuhui 838 and Luhui 602 decreased. NUEb of tested lines decreased by 2.7–21.2% and 0.8–17.4%,and NUEg decreased by 6.6–16.7% and 3.5–19.4% as nitrogen application levels increased from N0 to N90 and from N90 to N180,respectively.

3.2.Yield components of lines with different grain yields and NUEs

Tested varieties were divided into four types (Fig.1). HYHN included Yixiangyou 2115 and five restorer lines (Lehui 188,Shuhui 498,Luhui 17,Xikehui 768,and Fuhui 838);LYLN included six restorer lines (Duohui 1,Chenghui 3203,Chenghui 727,Yahui 2115,Minghui 63,and Shuhui 527);HYLN included Mianhui 725,Mianhui 523 and Luhui 602;and LYHN included Fuhui 718 only. Analysis of variance showed that number of panicles per unit area was the only component significantly affected by nitrogen application levels. SPP,grain setting rate and grain weight were significantly influenced by genetic factors in both years(Table 2). A comparison of yield components among lines with different grain yields and NUEs showed that Lehui 188 and Shuhui 498 in the HYHN group had significantly higher SPPs than the other lines. Among LYLN lines,Yahui 2115 generated SPPs less than 105 and 123 in 2015 and 2016,respectively. Moreover,Minghui 63 and Duohui 1 generated higher average numbers of panicles per unit area(＞250×104ha-1) in 2015.

Data of the 2 years showed that most HYHN rice plants displayed a medium or large panicle type. They had SPPs of 138.5 and 218.3 and average grain weights of 32.9 and 35.5 mg in 2015 and 2016,respectively. In contrast,LYLN plants displayed a small panicle type,with an average SPP of less than 123 for both years. Therefore,HYHN restorer lines had large SPPs and WPP. Correlation analyses were performed to determine the relationships between paniclecharacteristics,grain yield,and NUE (Fig.2). Grain yield was significantly and positively correlated with SPP and WPP (Fig.2-A and B),and SPP was significantly positively correlated with NUEg under N90 and N180 (Fig.2-C). A significant quadratic relationship was found between WPP and NUEg or NHI (Fig.2-D and F). Specifically,NUEg and NHI increased as WPP increased at low nitrogen levels(Fig.2-D and F). In addition,panicle biomass from heading to maturity was significantly positively correlated with NUEg(Fig.2-E). Although theR2values of regression models of NUEg with SPP,WPP and NHI with WPP were relatively low (0.11–0.17),indicating that the predictions were not accurate enough,they still demonstrated a statistically significant relationship between the independent and dependent variables.

Table 2 Yield and yield components of the tested varieties under different nitrogen applications in 2015 and 2016

(Continued on next page)

Table 2 (Continued from preceding page)

3.3.Nutrient transportation to the panicle in HYHN and LYLN restorer lines

Sink potential of population,determined by panicles per unit area,SPP and fulfilled grain weight,is the basis of yield formation (Donget al.2009b). In this study,both HYHN and LYLN groups contained varieties with high and low panicles per unit area,and only the effective panicles per unit area was significantly affected by nitrogen application amounts.Therefore,we paid more attention to the differences in SPP and grain weight that were the main components of the sink potential per stem. Meanwhile,we found that the HYHN restorer lines had large sink potential per stem owing to their large SPP and grain weight (Table 2);furthermore,SPP and WPP were significantly positively correlated with grain yield and NUE (Fig.2). Therefore,we inferred that large sink potential per stem helped HYHN restorer lines to obtain high grain yields and high NUEs. To explain how large sink potential per stem helped rice plants increased grain yield and NUE,we further explored the differences in nutrient transportation to the panicles between HYHN and LYLN restorer lines.

Accumulation and distribution of dry matter and nitrogen in the panicleAs depicted from Fig.3-A and B,dry matter weight ratio of panicle increased significantly from heading to maturity. During the heading stage,the HYHN restorer lines showed no advantage in dry matter weight ratio of panicle compared with their LYLN counterparts. However,at maturity,dry matter weight ratio of panicle in the HYHN restorer lines was significantly higher than that in the LYLN restorer lines under N0,N90 and N180 in both years,except for Duohui 1 under N90 in 2016. Similarly,NA of panicle increased with the developmental process after heading,and the HYHN restorer lines showed significantly higher NA of panicle than the LYLN restorer lines at maturity in 2016,except for Duohui 1 under N0. In addition,Shuhui 498 had significantly higher NA of panicle than LYLN restorer lines at maturity in 2015 (Fig.3-C and D). Therefore,compared with the LYLN restorer lines,the HYHN restorer lines showed advantages in the accumulation and distribution of dry matter and nitrogen in panicle from heading to maturity.

Vascular bundlesThe number of macro-vascular bundles and small-vascular bundles of neck-panicle nodes can reflect the size of the sink potential and whether the transportation of assimilates is smooth. Light microscopy images of cross-sections of neck-panicle nodes at the full-heading stage under the three nitrogen application treatments in 2016 are shown in Fig.4-A. Additionally,a comparison of the crosssectional area of neck-panicle nodes among the different strains showed the following trend:HYHN＞LYLN. The vascular tissue of neck-panicle node is composed of marco-vascular and small vascular bundles,and a vascular bundle is composed of phloem and xylem (Fig.5). The numbers of macro-vascular and small vascular bundles in Shuhui 498 (an HYHN restorer line) were significantly higher by 33.9 and 25.5%,respectively,than those of Yahui 2115 and by 43.5 and 60.9%,respectively,than those of Duohui 1. Similarly,those of Lehui 188 (an HYHN restorer line) were significantly higher by 24.5 and 7.1%,respectively,than those of Yahui 2115 and by 33.4 and 37.4%,respectively,than those of Duohui 1 (Table 4).Furthermore,the average value of the area of phloem and xylem of the macro-vascular bundles and small vascular bundles and the total area of the phloem and xylem,reflected the significant trend of HYHN＞LYLN,except for the area of the phloem of small vascular bundles for Yahui 2115. In addition,although Yahui 2115 and Lehui 188 had higher vascular bundle ratios than Shuhui 498 and Duohui 1,vascular bundle ratio of the HYHN restorer lines increased as nitrogen application increased,whereas that of Duohui 1 showed an opposite trend (Table 4).

Fig.1 Composite scores of factor analysis for yield (x) and nitrogen use efficiency (y) of the tested varieties in 2015. HY,high yield;LY,low yield;HN,high nitrogen-use efficiency(NUE);LN,low NUE.

Fig.2 Correlation between grain yield and spikelets per panicle (A) and weight per panicle (B);between nitrogen-use efficency for grain production (NUEg) and spikelets per panicle (C),weight per panicle (D) and panicle biomass from heading to maturity(E);and between nitrogen harvest index (NHI) and weight per panicle (F). N0,N90 and N180,no nitrogen application,nitrogen application of 90 and 180 kg ha–1,respectively.

Leaf vascular bundle tissue is an important part of the plant vascular system. The cross-sectional areas of leaf midribs showed the trend of HYHN＞LYLN,and vascular bundles of leaf midribs in the HYHN restorer lines were significantly more developed than those in the LYLN restorer lines under N90 and N180 (Fig.4-B).

Fig.3 Dry matter weight ratio of panicle in heading and maturity in 2015 (A) and 2016 (B),and nitrogen absorption (NA) of panicle in heading and maturity in 2015 (C) and 2016 (D). N0,N90 and N180,no nitrogen application,nitrogen application of 90 and 180 kg ha–1,respectively. Bars mean SD (n=3),and values of bar charts with different lowercase letters in the same group were significantly different at P＜0.05.

Neck-panicle and root bleeding intensitiesThe neckpanicle node is the last channel from the stem to the spike of the rice plants. Breeding intensity of neck-panicle node and root can reflect rice plants’ source and sink condition and root activity to some extent,respectively. A downward trend in neck-panicle node and root bleeding intensities per stem was observed 0–30 days after full-heading (Fig.6).Except for the neck-panicle node bleeding intensity per stem at 30 days after full-heading,Shuhui 498,an HYHN restorer line,showed significantly higher neck-panicle node and root bleeding intensities per stem than the LYLN restorer lines under the three nitrogen application treatments. In addition,neck-panicle node bleeding intensity per stem under N0 and root bleeding intensity per stem on 10 days after full-heading under N90 and N180 of Lehui 188 were higher than those of LYLN restorer lines (Fig.6-A and B).

4.Discussion

4.1.Yields and NUEs of restorer lines in response to nitrogen application

The application of nitrogen fertilizer is an important contributor to increased yield (Panet al.2012);however,under a high nitrogen application,the nitrogen absorbed by plants to produce a unit of grain yield increases,while NUE decreases (Zhanget al.2003;Panet al.2012;Conget al.2015).

Previous studies confirmed that yield and NUE of hybrid rice can be improved by reducing the amount of nitrogen applied. Indeed,hybrid rice shows high yields and NUEs under a low nitrogen application level of 100 kg ha-1(Wuet al.2016). Penget al.(2006) reported that yield and NUE of hybrid rice increased slightly with a 30% decrease in nitrogen application using modified fertilizer practices.This study showed that restorer lines responded differently to nitrogen application levels in terms of yield and NUE.Most restorer lines under N0-–N90 showed increased yields(8.25–34.62%) compared with those under N90–N180(–6.51–24.35%). However,some restorer lines,such as Luhui 602 and Mianhui 523,showed high grain yields with low nitrogen application and decreased yields under N90–N180 because of a significant decrease in SPP despite of an increased panicle number per unit area. In contrast,Zhanget al.(2010) and Zhang Set al.(2017) reported no significant increase in crop yield when nitrogen application was greater than 225 kg ha-1,although grain weight and SPP decreased at this concentration. Therefore,for some varieties,high nitrogen application reduced SPP and thus decreased yield. In addition,the present study showed that for restorer lines,only panicle number per unit area was significantly increased with the increase in nitrogen application. Accordingly,within a normal application range,nitrogen application generally increased the panicle number per unit area and thus increased rice yield.

Fig.4 Observation of vascular bundle of neck-panicle-nodes (A) and leaf midrib (B) in different restorer lines. N0,N90 and N180,no nitrogen application,nitrogen application of 90 and 180 kg ha–1,respectively. A,magnification times,40×;measuring scale,500 μm. B,magnification times,120×;measuring scale,200 μm.

Conversely,ANUE and PNUE of the restorer lines generally decreased as nitrogen application increased,consistent with a report by Zhanget al.(2010) describing hybrid rice. However,different restorer lines had different responses. For example,ANUE and PNUE of Lehui 188 and Chenghui 727 increased as nitrogen application increased,but those of other restorer lines deceased. Furthermore,as nitrogen application increased,NUEb and NUEg decreased,whereas TNA increased. In addition,basic soil fertility of paddy fields may also influence rice grain yield and NUE(Hossainet al.2005;Zenget al.2012). Therefore,different restorer lines had different optimum nitrogen applications to obtain high grain yields and NUEs. These findings showed that determining the optimum nitrogen application in terms of NAGof a variety and basal nitrogen supply of paddy field soil was important for rice efficient cultivation.

4.2.Relationship of sink potential with grain yield and NUE

Several researchers have studied sink potential of hybrid rices. For example,Chenet al.(2017) found that sink potential had a large influence on efficient absorption of nitrogen,and sink potential per unit area and spikelet number per unit area of high NUE rice were higher than those of low NUE rice. Furthermore,sink potential of large panicle varieties was found greater than that of small panicle varieties,and increase in panicle weight was beneficial to sink potential (Donget al.2009a). In the 1990s,Zhouet al.(1997) proposed the breeding theory of“intersubspecific heavy panicle hybrid rice”,which was based on certain panicle and grain setting rate and on increasing SPP to achieve higher yields. Zhanget al.(2009),Yanet al.(2014) and Tianet al.(2016) also confirmed that a large SPP was beneficial for yield. Moreover,large panicle-type rice has been shown to have a higher potential for nitrogen accumulation (Piaoet al.2004;Qinet al.2014). In this study,we showed that SPP and WPP were significantly positively correlated with grain yield and NUEg,within certain limits.Thus,sink potential has a close relationship with grain yield and NUE,and a large and heavy panicle could increase sink potential and is thus beneficial for both yield and NUE.

Fig.5 Distribution of macro-vascular bundle and small vascular bundle in the neck-panicle node,distribution of phloem and xylem in vascular bundle. Magnification times,120×;measuring scale,200 μm.

Studies on hybrid rice by Fenget al.(2014) and Liet al.(2015) revealed that HYHN hybrid rice has 185–198 spikelets and a panicle number per unit area of 268×104–286×104ha-1. In this study,we compared the HYHN and LYLN restorer lines and found that most of the HYHN restorer lines showed a medium or large panicle type. In terms of yield components,the HYHN restorer lines had an average SPP of 138.5 and 218.3 in two tested years,respectively;in contrast,the LYLN restorer lines showed a small panicle type,with an average SPP of less than 123 in both years. Therefore,the HYHN restorer lines had the advantage in terms of sink potential per stem;this wasconsistent with the findings of Zhanget al.(2006),who reported that sink potential of the HYHN rice was 25%greater than that of the LYLN rice. Furthermore,the HYHN restorer lines also had large grain weight. Maet al.(2003)and Yanet al.(2014) showed that heavy-panicle hybrid rice had a strong ability to assimilate CO2and maintain a high net photosynthetic rate. Thus,a high level of sink potential may contribute to high yield and NUE of the HYHN restorer lines. In addition,although there was no consistent rule of panicle number per unit area among different yields and NUE varieties,nitrogen application can be adjusted to increase the effective panicle number per unit area,so as to increase sink potential of population.

Table 4 Characters of the vascular bundle of neck-panicle nodes of rice in 2016

Fig.6 Neck-panicle node (A) and root (B) bleeding intensity per stem after full-heading in 2016. N0,N90 and N180,no nitrogen application,nitrogen application of 90 and 180 kg ha–1,respectively. Bars mean SD (n=3),and values of bar charts with different lowercase letters in the same group were significantly different at P＜0.05.

The contribution rates of restorer lines to yield-related traits were over 50%;in particular,the contribution rates of single plant yield and panicle weight were 81.80 and 73.23%,respectively (Zhouet al.2015). Zhang Jet al.(2017)reported that at low and high nitrogen levels,the effects of restorer lines on yield traits of hybrid rice were greater than that of sterile lines. In addition,Zhonget al.(2011)showed that branch and spikelet differentiation of hybrid rice was dramatically affected by restorer lines. Therefore,breeding of restorer lines with good panicle characteristics is important to produce HYHN hybrid combinations. In the current study,Lehui 188 was selected as an ideal HYHN restorer line. Zhouet al.(2015) found that Lehui 188 had remarkable potential as a restorer line inindicahybrid rice because of its strong combining and restoring ability.Furthermore,Fyou 498,a hybrid combination of Shuhui 498,has been confirmed as an HYHN hybrid rice (Tianet al.2016;Zhang Set al.2017). Therefore,five HYHN restorer lines -Lehui 188,Shuhui 498,Luhui 17,Xikehui 768,and Fuhui 838 -can be further applied for breeding of HYHN combination.

4.3.Advantages of nutrient transportation to the panicle in HYHN restorer lines

We found that the HYHN restorer lines had an advantage of large sink potential because of large SPP and WPP that were both closely related to yield and NUE;therefore,we further examined the differences in nutrient transportation to panicle between the HYHN and LYLN restorer lines. Jiet al.(2012)reported that the peak of dry matter accumulation in HYHN rice occurred from heading to maturity. We demonstrated that panicle biomass during this period was significantly positively correlated with NUEg. Therefore,transportation of dry matter to panicle after heading is important to increase yield and NUE. We found that the HYHN restorer lines showed advantages in accumulation and distribution of dry matters and nitrogens in panicle from heading to maturity compared with the LYLN restorer lines. Weiet al.(2008)also reported that nitrogen-efficient rice accumulated a large amount of nitrogen and transported it to grains after heading.In addition,NA from heading to maturity contributed 8.77 and 15.90% to NUEg and NHI,respectively (Jiet al.2012.Therefore,the HYHN restorer lines with large sink potentials showed advantages in accumulation and distribution of dry matter and nitrogen in panicles,providing a basis for high yield and NUE.

We further studied the characteristics and differences in nutrient transportation pathways of the HYHN and LYLN restorer lines,from root to ground and from stem and leaf to spike,respectively. We found that the HYHN restorer lines had a higher root bleeding intensity per stem after heading,consistent with the results of Liet al.(2012);furthermore,amino acid concentration of bleeding sap in high-NUE rice was higher than that of low-NUE rice (Zhang 2006). In addition,HYHN rice might have highly developed roots. Liet al.(2012) confirmed that HYHN rice has advantages such as high root dry weight,root volume,root total absorption surface area,and active absorption surface area at each growth stage (Liet al.2012);similar conclusions were obtained for high-NUE rice,but not for low-NUE rice,after jointing (Jiet al.2012). Therefore,the HYHN restorer lines may have advantages in transporting nutrients from roots to ground.

Neck-panicle node is the last passage of photosynthates traveling from stem to spike. Phloem mainly transports photosynthates and,together with xylem that transports water,forms vascular system. We found that the area of phloem and xylem in macro-and small-vascular bundles of neck-panicle nodes of the HYHN restorer lines was significantly higher than that of the LYLN restorer lines.Moreover,vascular bundle ratio of the HYHN restorer lines increased as nitrogen application increased. Therefore,we concluded that the vascular bundle tissues of neck-panicle node of the HYHN restorer lines developed and exhibited a positive response to nitrogen application. Moreover,the HYHN restorer line Shuhui 498 showed a significantly higher bleeding intensity per stem of neck-panicle node than the LYLN restorer lines after full heading. The HYHN restorer lines also exhibited well-developed leaf vascular bundles.Therefore,the HYHN restorer lines may have advantages in transporting nutrients from stem to spike and from leaf to stem. Additionally,we found that Yahui 2115,a LYLN restorer line,had a significantly lower phloem and xylem area in neck-panicle node than the HYHN restorer lines,though it had a strong stem and high vascular bundle ratio.Furthermore,particularly under N0,the neck-panicle node bleeding intensity of Yahui 2115 after full heading was significantly lower than that of the HYHN restorer lines,suggesting that nutrient transportation between stem and spike was limited in this line,possibly because of its small spike.

5.Conclusion

The present study on NUE ofindicahybrid restorer lines in Southwest China showed that grain yield and NUE of different restorer lines responded differently to nitrogen application;the HYHN restorer lines had a larger sink potential and higher capacity to transport assimilates than the LYLN lines,therefore yielding higher level of assimilate production to obtain a high yield while increasing the use of nitrogen.

Acknowledgements

This work was financially supported by grants from the National Natural Science Foundation of China (31871564),the National Key R&D Program of China (2016YFD0300506 and 2017YFD0301700) and the Sichuan Breeding Key Research Project,China (2016NYZ0051). We thank Prof.Huang Fu from the College of Agronomy,Sichuan Agricultural University,for providing the experimental materials,and the students and farmers for their help with the experiments.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm