Tiller fertility is critical for improving grain yield,photosynthesis,and nitrogen efficiency in wheat

DING Yong-gang,ZHANG Xin-bo,MA Quan,LI Fu-jian,TAO Rong-rong,ZHU Min,,LI Chun-yan,,ZHU Xin-kai,,GUO Wen-shan,,DING Jin-feng,,

1 Jiangsu Key Laboratory of Crop Genetics and Physiology/Jiangsu Key Laboratory of Crop Cultivation and Physiology/Agricultural College,Yangzhou University,Yangzhou 225009,P.R.China

2 Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops,Yangzhou University,Yangzhou 225009,P.R.China

3 Jiangsu Ruihua Agricultural Technology Co.,Ltd.,Suqian 223800,P.R.China

Abstract Genetic improvement has promoted wheat’s grain yield and nitrogen use efficiency (NUE) during the past decades.Therefore,the current wheat cultivars exhibit higher grain yield and NUE than previous cultivars in the Yangtze River Basin,China since the 2000s. However,the critical traits and mechanisms of the increased grain yield and NUE remain unknown. This study explores the mechanisms underlying these new cultivars’ increased grain yield and NUE by studying 21 local cultivars cultivated for three growing seasons from 2016 to 2019. Significantly positive correlations were observed between grain yield and NUE in the three years. The cultivars were grouped into high (HH),medium(MM),and low (LL) grain yield and NUE groups. The HH group exhibited significantly high grain yield and NUE. High grain yield was attributed to more effective ears by high tiller fertility and greater single-spike yield by increasing postanthesis single-stem biomass. Compared to other groups,the HH group demonstrated a longer leaf stay-green ability and a greater flag leaf photosynthetic rate after anthesis. It also showed higher N accumulation at pre-anthesis,which contributed to increasing N accumulation per stem,including stem and leaf sheath,leaf blade,and unit leaf area at preanthesis,and promoting N uptake efficiency,the main contribution of high NUE. Moreover,tiller fertility was positively related to N accumulation per stem,N accumulation per unit leaf area,leaf stay-green ability,and flag leaf photosynthetic rate,which indicates that improving tiller fertility promoted N uptake,leaf N accumulation,and photosynthetic ability,thereby achieving synchronous improvements in grain yield and NUE. Therefore,tiller fertility is proposed as an important kernel indicator that can be used in the breeding and management of cultivars to improve agricultural efficiency and sustainability.

Keywords: grain yield,NUE,tiller fertility,photosynthesis,nitrogen uptake

1.Introduction

Food production needs to increase by 70-100% by 2050 to meet global food demands (Godfrayet al.2010). Wheat is a valuable and widely cultivated crop that provides around 19% of the calories and 21% of the protein for human diets globally (FAO 2019). Therefore,increasing wheat yield will help resolve food shortage issues. Wheat yield improvement has been studied from various perspectives,including planting pattern(Duet al.2021b),tillage (Brennanet al.2014),irrigation(Shahrokhnia and Sepaskhah 2016),chemical fertilizer(Junet al.2017),gene mining (Rebetzke et al.2011),and plant protection (Curtiset al.2012). Nitrogen (N) is a crucial element driving plant growth that directly affects leaf area,leaf emergence rate,photosynthetic capacity,and radiation interception (Tafteh and Sepaskhah 2012;Shahrokhnia and Sepaskhah 2016). In the 1960s,crop yield was significantly increased by increasing N fertilizer application,and this fertilization approach has since been widely recommended in wheat production (Sinclair and Rufty 2012). However,further increases in N application in the 2000s did not result in any further increases in grain yield (Vitouseket al.2009). Excessive fertilizer loss is not only economically costly but also has caused air and water pollution (Hawkesfordet al.2014;Duanet al.2019).Improving the N use efficiency (NUE) while gaining as much yield as possible with appropriate N input has been proposed as a solution to this issue (Hawkesfordet al.2014).

NUE includes N uptake efficiency (NUpE) and N utilization efficiency (NUtE) (Rasmussenet al.2015).NUpE is defined as the percentage of the extra applied N harvested in the plant (Rasmussenet al.2015). To promote NUpE,some studies have optimized the amount,ratio,period,and types of N fertilizer according to the requirements of N during the wheat growth period (Duanet al.2019;Maet al.2021). NUpE is also related to the N uptake capacity of wheat,which is mainly related to the root morphology,nitrogen transporter gene expression,xylem and phloem loading,and translocation (Kantet al.2018;Sinhaet al.2020). The NUtE has been estimated from the grain yield increase per unit of incremental N accumulation (Cassmanet al.2003) and is a complex physiological process that includes N assimilation,signaling,and remobilization,which control plant photosynthesis,leaf expansion,and growth (Zhanget al.2019;Huet al.2018;Kantet al.2018). NUtE reflects the capacity for N to contribute to grain yield production (Tianet al.2015).

Previous studies have reported that increasing the N uptake was conducive to the photosynthetic system development,such as chloroplast,amino acid,and enzyme,more photosynthetic assimilation transport from the green organs into the grains,and then increased grain yield (Duanet al.2019;Yinet al.2019;Neheet al.2020).However,excess N is stored in the vacuoles and does not participate in N assimilation because of the threshold limit of N utilization (Wanget al.2012). Improving the N utilization threshold is thus a key target for further enhancing yield potential in Argentina (Cavigliaet al.2014). In Iran,the grain yield of wheat was also increased by synchronously promoting the potential N uptake and utilization efficiency (Fatholahiet al.2020). These results imply that the mechanisms of grain yield and NUE differ and are dependent on the production region. Therefore,clarifying this mechanism could inform fertilizer application and promote yield improvement to realize the sustainable regional production of wheat.

The Yangtze River Basin is an important wheat production region in China,constituting 16% of the total wheat production area and accounting for 25% of total production (Tianet al.2011). In this region,genetic improvements have synchronously increased the grain yield,NUpE,and NUtE of wheat in recent decades (Tianet al.2016). However,the agronomic traits of the highgrain yield and high-NUE cultivars that were released in recent years have not been characterized,and the mechanisms by which higher grain yield and NUE levels have been achieved are not known. In the present study,21 weak-winter wheat cultivars (TriticumaestivumL.) that were released in the Yangtze River Basin after 2000 were cultivated for three seasons from 2016 to 2019 to: (1)identify the relationship between the grain yield and NUE of different wheat cultivars;(2) explore the differences in yield components,NUtE,NUpE,single-stem biomass,nitrogen accumulation,and allocation among different cultivars;and (3) determine the essential traits and mechanisms for breeding new high-grain yield and high-NUE cultivars.

2.Materials and methods

2.1.Site conditions

The field experiments were conducted at the farm of Yangzhou University (32°23′N,119°25′E) during the wheat seasons of 2016-2017 (2017),2017-2018 (2018),and 2018-2019 (2019). A wheat-rice rotation system was used. The soil type of the experiment was clay loam.The soil nutrient contents in the 0-20 cm soil layer before sowing are shown in Table 1. The precipitation and daily mean temperature in the wheat-growing seasons are given in Fig.1.

Table 1 Soil nutrient contents in experimental fields during the wheat growth seasons of 2016-2017 (2017),2017-2018(2018),and 2018-2019 (2019)

2.2.Experimental design and field management

Twenty-one wheat cultivars widely cultivated in the Yangtze River Basin and released in the 2000s and2010s were selected. According to the recording of China National Seed Association (CNSA 2016),six cultivars among the tested cultivars that were released before 2010 were sown on over 6 million hectares,and 10 cultivars that were released from 2010 to 2013 were sown on approximately 50 thousand hectares after the growing seasons of 2017. The year of release and pedigree of these cultivars are shown in Table 2.

Table 2 The year of release and pedigree of the cultivars planted in Yangzhou,China

All cultivars were used in a randomized complete block design with three replications. Each plot size was 3 m×3 m.The seeds were planted at a depth of approximately 3 cm in the topsoil with a 0.25-m line spacing. The seedlings were removed or transplanted to achieve a planting density of 225 plants m-2at the three-leaf stage (Zadoks stage,GS13). The N ratio was 120,24,48,and 48 kg ha-1applied at the stages of pre-sowing,four-leaf (GS14),stem elongation (GS32),and booting (GS45),respectively.Potassium (K2O) and phosphorus (P2O5) were applied twice at 60 kg ha-1at the pre-sowing and stem elongation stages. To calculate the use efficiency of N fertilizer,control plots without N input were separately sown with the tested cultivars. The sowing dates were 16 November 2016 and 30 October 2017 and 2018. The sowing date was delayed in 2016 due to the extreme precipitation in October (Fig.1). Pests,weeds,and diseases prevention was conducted routinely,and there was no irrigation management in the three years due to abundant rainfall.According to our investigation,the difference in the date of the stem elongation (GS32),anthesis (GS65),and maturity (GS92) stages among the cultivars was within 3,3,and 1 day,respectively.

Fig. 1 Precipitation and daily mean temperature in the experimental site during the wheat growth seasons of 2016-2017 (2017),2017-2018 (2018),and 2018-2019 (2019).

Fig. 2 Dendrogram of nitrogen use efficiency (NUE) and grain yield of wheat cultivars using Ward’s clustering algorithm with Euclidean distances in 2017,2018,and 2019. ---indicates the threshold,and all cultivars left of the line were grouped as the same type according to the Euclidean distance of 7.0.

Fig. 3 Correlations between nitrogen use efficiency (NUE) and grain yield of different wheat cultivars in 2017,2018,and 2019.＊＊ indicates significance at P＜0.01.

Fig. 4 Maximum stem and tiller number (A),tiller fertility (B),single-stem biomass at pre-anthesis (C) and post-anthesis (D) of different cultivar groups in 2017-2019. Bars mean SD (n=3). Different lowercase letters on the bars indicate significant differences among cultivar groups at P＜0.05.

Fig. 5 Correlations of tiller fertility of different wheat cultivars with the stay-green integral (A),flag leaf net photosynthetic rate (Pn)at anthesis (B) and milk-ripe stage (C),N accumulation per stem (D),leaf N accumulation per stem (E),and N accumulation per unit leaf area (F) in 2017,2018,and 2019. ＊＊ and ns indicate significance at P＜0.01 and no significance,respectively.

2.3.Crop measurements

Grain yield and yield componentsAt the maturity stage (GS92),an area of 1 m2within the plot from the center of each plot was selected to record the effective ears,following which the wheat plants were manually harvested,threshed,and weighed. The grain moisture was measured by a Grain Analyzer (Infratec? 1241,Foss,Denmark). One thousand grains were randomly selected from harvested grain by hand and weighed as thousand-grain weight. The grain yield and thousandgrain weight were adjusted to 13% moisture. Fifty spikes in the middle of each plot were sampled at the milk-ripe stage (GS75) to measure grains per ear. The single-spike yield was calculated as the single-grain weight multiplied by the grains per ear. The single-grain weight was calculated by dividing the thousand-grain weight by 1 000.

Stem and tiller number and tiller fertilityAn area of 0.75 m2in each plot was selected to record the number of stem and tiller at the stem elongation (GS32) and maturity (GS92)stages;tiller＞2 cm in length were recorded. Stem and tiller number at the elongation stage represented the maximum stem and tiller number. Tiller fertility was calculated by dividing the fertile tiller number by the maximum tiller number. The fertile tiller number was the difference between the effective ears and the main stem number.

Biomass,leaf area,and net photosynthetic rate (Pn)At the anthesis (GS65) and milk-ripe (GS75) stages,20 wheat plants were harvested from each plot. The samples were separated into green leaf laminae,stem and leaf sheath,yellow leaf laminae (exceeding half of the lamina was yellow),and spike at the anthesis and milk-ripe stages.Green leaf area was measured with an LI-3000 (LI-COR Inc.,Nebraska,USA). At the maturity (GS92) stage,the samples were separated into leaf laminae,stem and leaf sheath,and spike. The stay-green integral was used to evaluate the ability of overall green leaf area retention of the crop after anthesis and was calculated as the integral from the anthesis to the milk-ripe stage. It corresponded to the area under the stay-green curve from the anthesis to the milk-ripe stage (Christopheret al.2018).

where L1 and L2 represent the leaf area per stem at the anthesis and milk-ripe stages,respectively,D1 and D2 represent the date of the anthesis and milk-ripe stages,respectively.

All samples were dried at 80°C to a constant weight and then weighed. The biomass at anthesis was represented as biomass at pre-anthesis. The postanthesis biomass represented the increase in biomass from the anthesis to the maturity stage.

At the anthesis and milk-ripe stages,thePnof five flag leaves from each plot was measured from 9:30 to 11:30 a.m.on a sunny day using a portable photosynthesis device(LI-6400,LI-COR Inc.,USA). The chamber was equipped with a red/blue LED light source (LI-6400-02B).The environmental conditions of the chamber during measurement were according to Muet al.(2010).

Nitrogen accumulation and NUEThe dried samples that were sampled at anthesis and maturity were milled and analyzed for N concentration using the Kjeldahl method reported by Santos and Boiteux (2013). The NUE,NUpE,and NUtE were calculated according to the methods of Rasmussenet al.(2015) and Cassmanet al.(2003).

Nitrogen accumulation (kg ha-1)=N content×Biomass (2)

Nitrogen use efficiency (NUE,kg kg-1)=

Nitrogen uptake efficiency (NUpE,%)=

Nitrogen utilization efficiency (NUtE,kg kg-1)=

where Y1 and Y0 are the grain yield under N and control treatments,respectively. T1 and T0 represent the N accumulation of plants under N and control treatments,respectively.

2.4.Statistical analysis

According to the grain yield and NUE of cultivars in the three growing seasons from 2016-2019 (Table 3),we classified these tested cultivars into three groups for each year,including the high-yield and high-NUE group(HH),medium-yield and medium-NUE group (MM),and low-yield and low-NUE group (LL),using hierarchical cluster analysis (Ward’s method and Euclidean distances)(Fig.2). All data were analyzed with one-way ANOVA in SPSS 19.0 (SPSS,Inc.,Chicago,IL,USA) to compare the differences in grain yield,NUE,single-stem biomass,and nitrogen accumulation and allocation among cultivar groups. Multilinear regression was used to analyze the contribution of yield components to grain yield by SPSS.Significant differences were assessed using at-test.Pearson’s correlation analysis was carried out atP＜0.05.The data of the HH,MM,and LL groups were expressed as the mean±standard error.

Table 3 Nitrogen use efficiency (NUE) and grain yield of wheat cultivars in the wheat growth seasons of 2016-2017 (2017),2017-2018 (2018),and 2018-2019 (2019)1)

3.Results

3.1.Grain yield and NUE

The grain yield and NUE of HH were significantly higher(by 15 and 18%,14 and 23%,and 12 and 20% in 2017,2018,and 2019,respectively) than those of MM,and MM was significantly higher (by 14 and 22%,11 and 34%,and 23 and 55% in 2017,2018,and 2019,respectively) than LL (Table 4). NUE was significantly positively correlated with grain yield in the three years (Fig.3).

Table 4 Grain yield and nitrogen use efficiency (NUE) of different cultivar groups in 2017,2018,and 2019

3.2.Yield components

As shown in Table 5,the effective ears of HH were significantly higher than that of MM in three years,and MM was greater than LL,except for 2017. There was no significant difference in the number of grains per ear among the three cultivar groups,with the exception that HH and MM yielded noticeably more grains per ear than LL in 2019. Thousand-grain weight of HH was greater than in LL,though no significant difference was detected among groups in 2019. The HH group had the highest single-spike yield among the cultivar groups,followed by MM and LL. The multilinear regression analysis of yield components showed that effective ears and singlespike yield were the major factors influencing grain yield(Table 6).

Table 5 Yield components of different cultivar groups in 2017,2018,and 2019

Table 6 Contributions of yield components for grain yield by the multilinear regression analysis in 2017,2018,and 2019

3.3.Tillering ability and single-stem biomass

As shown in Fig.4,the tiller fertility of HH was significantly higher than that of MM in three years,and MM was significantly higher than LL in 2018 and 2019. Therewas no significant difference in maximum stem and tiller number among the HH,MM,and LL groups in the three years,except for 2017,when this value of HH was significantly higher than MM and LL.

The single-stem biomass at pre-anthesis was similaramong HH,MM,and LL,with the exception that HH was significantly greater than the other groups in 2018.Compared with MM,the single-stem biomass at postanthesis of HH was significantly higher by 14% (2017),11% (2018),and 16% (2019),and that of LL was significantly lower by 12,7,and 6%.

3.4.Leaf area per stem and flag leaf Pn

As shown in Table 7,there was no significant difference in leaf area per stem at anthesis among the three cultivar groups in three years. The stay-green integral of HH was greater than in LL and only differed significantly between HH and MM in 2019. The HH group showed the highest flag leafPnvalue at the anthesis and milkripe stages compared with the other groups,though no significant difference was detected among the groups in 2018. The flag leafPnvalues between MM and LL were similar,with the exception that thePnat anthesis of MM was greater than that of LL in 2019. These findings imply that a high leaf photosynthetic rate and low photosynthetic area attenuation are key to achieving high photosynthesis.

Table 7 Leaf area,stay-green integral,and flag leaf net photosynthetic rate (Pn)at anthesis and milk-ripe of different cultivar groups in 2017,2018,and 2019

3.5.NUpE,NUtE,and nitrogen accumulation

Compared with MM,the NUpE of HH was higher by 26,18,and 17% in 2017,2018,and 2019,respectively,and LL was lower by 14% (2017),15% (2018),and 32% (2019)(Table 8). The difference in NUtE was not significant among HH,MM,and LL in the three years. Compared with MM,N accumulation at anthesis in HH was higher by 26,15,and 14% in 2017,2018,and 2019,respectively,and LL was lower by 14% (2017),15% (2018),and 17%(2019). The difference in N accumulation at post-anthesis was not significant among the cultivar groups (Table 7).The present results indicate that high N uptake capacity contributes to high crop productivity and N efficiency,particularly before flowering.

Table 8 Nitrogen uptake efficiency (NUpE),nitrogen utilization efficiency (NUtE),and N accumulation at pre-anthesis and post-anthesis among different cultivar groups in 2017,2018,and 2019

3.6.Nitrogen allocation to the organs

As shown in Table 9,HH had significantly higher N accumulation per stem and leaf N accumulation per stem than MM,followed by LL. Stem and leaf sheath N accumulation per stem of HH was significantly higher than that of LL. The difference in stem and leaf sheath N accumulation per stem was not significant between HH and MM or MM and LL,although the difference was significant among HH,MM,and LL in 2017. The difference in spike N accumulation per stem was not significant among the cultivar groups. Compared with MM,N accumulation per unit leaf area of HH was higher by 18,18,and 12% in 2017,2018,and 2019,respectively,and it decreased by 11% (2017),14% (2018),and 19%(2019) in LL.

Table 9 Nitrogen accumulation per stem,leaf,stem and leaf sheath,and spike N accumulation per stem and N accumulation per unit leaf area at anthesis of different cultivar groups in 2017,2018,and 2019

3.7.Relationships between tiller fertility and staygreen integral,flag leaf Pn,and N accumulation

Tiller fertility was significantly positively correlated with the stay-green integral,flag leafPnat anthesis and postanthesis,N accumulation per stem,leaf N accumulation per stem,and N accumulation per unit leaf area,though only a weak relationship was observed between tiller fertility and flag leafPnat anthesis in 2018 (Fig.5).

4.Discussion

4.1.Synergistic analysis of grain yield and NUE among wheat cultivars

Nitrogen is a key nutrient for wheat growth,and increasing N input notably promotes grain yield but inevitably reduces NUE (Hawkesfordet al.2014;Duanet al.2019). The NUE potential also differs significantly among genotypes,and thus choosing genotypes with high NUE can potentially help reduce the N application rate without yield loss (Tianet al.2016;Cohanet al.2019). In this study,a positive correlation was detected between grain yield and NUE across three years (Fig.3). The grain yield and NUE of HH were significantly higher than that of MM,followed by LL in three years (Table 4),indicating that the grain yield and NUE of wheat cultivars that were released after 2000 were increased synchronously in the Yangtze River Basin. Moreover,the grain yield and NUE of the same cultivars had different levels in different years(Table 3),indicating the variances of cultivars adapting sowing date (late sowing in 2017),soil moisture condition(excessive precipitation in the winter in 2017 and 2019),and different growth environments.

4.2.Grain yield formation mechanism of cultivars with high yield and high NUE

Growing region significantly affects the contributions of effective ears,grains per spike,and thousand-grain weight to crop grain yield (Tianet al.2011). In the North China Plain and Mediterranean Basin,the contribution of effective ears to grain yield (from 2 to 7 t ha-1) was found to be greater than that of grains per ear and thousandgrain yield;nevertheless,the decreased grains per ear and thousand-grain yield were offset by the improvementin effective ears to grain yield (about 9 t ha-1) (Luet al.2015;Subiraet al.2015;Zhenget al.2021). In the Yangtze River Basin,increased grain yield was mainly attributed to high single-spike yield with genetic improvement of wheat (Tianet al.2011). The yield components were also affected by precipitation. Increased precipitation decreased the tillering capacity at the seedling stage and then reduced effective ears. Furthermore,it also decreased the number of grains per ear and thousand-grain weight because of photosynthesis weakening at the grain-filling stage (Cuiet al.2015;Duet al.2021a). In this study,there was higher precipitation at the seedling stage(from October to December) in 2017 and 2019 than in 2018,which reduced the effective ears in 2017 and 2019. The grains per ear and thousand-grain weight were reduced due to more precipitation in May 2018 (Table 5). The effective ears and single-spike yield were still the main factors for grain yield in three years(Table 6). Effective ears and single-spike yield of HH were significantly higher than that of MM,followed by LL (Fig.4).These results suggest that increases in both effective ears and single-spike yield are characteristic of high-yield cultivars(7 t ha-1) in the Yangtze River Basin.

Improving tillering increased the population size and use efficiency of various resources,thereby promoting final effective ears and grain yield (Liuet al.2020). A suitable N application could lower the ineffective tiller number and obtain enough tillers,and excessive N input increased not only the tiller number but also the ineffective tiller number,possibly resulting in aggravated competition among individuals and a negative effect on effective ears (Ishag and Taha 1974;Berryet al.2003;Luet al.2021;Yanget al.2021). In this experiment,there was no significant difference in the maximum stem and tiller number among the cultivars in an appropriate N rate supply. The HH group had the highest tiller fertility,followed by MM and LL (Fig.4),suggesting that the effective ears of the high-yield cultivars were increased by high tiller fertility but not by the maximum stem and tiller number. The tillers were fewer in 2017 and 2019 than in 2018 due to excessive precipitation in the winter of 2017 and 2019. The reason was that excessive precipitation at the seedling stage reduced the growth of tillers and inhibited tillering (Collaku and Harrison 2002;Yanget al.2021). Although these adversity conditions resulted in low tillering ability,the strong recovery capacity of wheat cultivars improved tiller survival to increase the number of productive ears (Sundgrenet al.2015). Therefore,cultivars exhibit distinct adaptability in response to specific adversities,resulting in different tiller fertility of the same cultivars under different ecological conditions.This highlights the need to evaluate varieties in multiple environments.

Carbohydrate produced by photosynthesis at postanthesis contributes 60-90% to the final single-spike yield.Therefore,photosynthetic production at post-anthesis determines the final single-spike yield and grain yield(Masoniet al.2007;Dordaset al.2012). In this study,the difference in single-stem biomass at pre-anthesis was not significant among the cultivar groups. The single-stem biomass at post-anthesis of HH was the highest,followed by MM and LL (Fig.4). This indicated that greater singlestem biomass at post-anthesis promoted single-spike yield in the high-yield cultivars.

The leaf is the main photosynthetic organ of crops,and large leaf area is tightly related to biomass and grain yield by increasing the amount of intercepted radiation and the energy transformation capacity (Taoet al.2018). Flag leaves capture most of the energy compared with other leaves,and flag-leaf photosynthesis contributes 30-50%of grain-filling assimilation in wheat (Blandino and Reyneri 2009;Guoet al.2015). The photosynthetic production of leaves declines,resulting from leaf senescence,and the photosynthetic apparatus breaks down (Brouweret al.2012). Therefore,leaf area and flag leaf photosynthesis maintain at a high level after anthesis play an important role in wheat yield formation (Xuet al.2016). In this study,leaf area per stem at anthesis was not significantly different among the cultivar groups. The stay-green integral and flag leafPnof HH were significantly higher than that of MM and LL (Table 7). These results indicated that the high-yield cultivars had a longer leaf stay-green ability and flag leaf photosynthetic potential.

4.3.Nitrogen utilization,uptake,and allocation characters of cultivars with high yield and high NUE

Increasing NUE (including NUpE,NUtE,or both) is an important method for maintaining high crop productivity(Cassmanet al.2003;Duanet al.2019;Fatholahiet al.2020). The variation in NUE strategies among wheat cultivars can mainly be explained by genetic differences(Gajuet al.2011;Neheet al.2018). In this study,NUtE exhibited no significant difference among the cultivar groups. The NUpE of HH was significantly higher than that of MM and LL (Table 8). These results suggested that NUpE was the limiting factor for cultivars that were released after 2000 in the Yangtze River Basin. NUE,NUpE,and NUtE of cultivars were higher in 2019 than in 2017 and 2018,likely due to relatively appropriate rainfall during the medium and late growth period in 2019. High rainfall in 2017 and 2018 was probable to have limited N uptake and utilization through weakening root absorption ability,reducing N transport,and decreasing leaf photosynthetic capacity (Wanget al.2015;Xuet al.2016). NUpE is mainly determined by N accumulation at pre-anthesis in plants because strong root physiological activities promote N acquisition from the soil during the vegetative period,and these activities limit N uptake during the reproductive period (Sinclairet al.2012;Huet al.2018). In this experiment,the N accumulation of HH was significantly higher than that of MM at pre-anthesis,followed by LL,and the difference in N accumulation at post-anthesis was not significant among the cultivar groups (Table 8). These results indicated that the high-NUE cultivars had high NUpE,which was mainly related to N accumulation at preanthesis.

Although the N accumulation of individual plants is diluted by higher numbers of plants per unit land area for a given N uptake,improving NUpE promotes the N content of individual plants (Yinet al.2019). HH had significantly higher N accumulation per stem than MM at anthesis,and LL was the lowest (Table 9). Previous studies have shown that around 40,40,and 20% of the N accumulation per unit area at anthesis were allocated in the leaf lamina,stem and leaf sheath,and ear,respectively (Pasket al.2012;Gajuet al.2014). Leaf N content is an essential trait that is used to evaluate leaf photosynthesis and canopy senescence after anthesis (Yinet al.2019). A high level of N stored in the stem before anthesis could translocate more N to the grain without decreasing photosynthesis during grain filling (Foulkeset al.2009). In the present study,HH had the highest leaf,stem,and leaf sheath N accumulation per stem and N accumulation per unit leaf area,followed by MM and LL,though the difference in spike N accumulation per stem among the cultivars was not significant (Table 9).These results indicated that the N accumulation per stem of high-NUE cultivars was higher and increased the N allocation in the leaf,stem,and leaf sheath,as well as the per unit leaf area.

4.4.Tiller fertility was a critical trait for improving grain yield and NUE

Previous studies showed that tiller number was strongly synchronized with root number or biomass. The main reason was that the developed root absorbed more nitrogen to support tiller fertility (Ishaget al.1974;Klepperet al.1984;Allardet al.2013). In this study,there was a positive correlation between tiller fertility and N accumulation in the individual plant,leaf,and per unit leaf area (Fig.5). A high leaf N content promotes photosynthetic capacity (Rubisco,chlorophyll,stomatal conductance) and delays leaf senescence (Makinoet al.1997;Yinet al.2019). Herein,tiller fertility had a positive correlation with stay-green integral andPnat post-anthesis(Fig.5).

Tiller fertility was the link between NUE and grain yield among the wheat cultivars. Improving tiller fertility increased the N absorption per stem before anthesis,likely through optimizing root morphology (Huet al.2018;Sinhaet al.2020). This enhanced the N allocation level in the leaf to delay the leaf senescent at post-anthesis,which was beneficial to more photosynthetic assimilation transported into grains. In addition,high tiller fertility also directly increased the effective ears (Fig.6). The characteristics of tiller fertility,root morphology,and their connection to high-yield and -NUE of wheat will be our research direction in the future.

Fig.6 Structural modeling of nitrogen use efficiency (NUE),grain yield,and agronomic traits. Details of the correlations are shown in Appendices A-H and Fig.4. NUtE,nitrogen utilization efficiency;NUpE,nitrogen uptake efficiency;NAPA,nitrogen accumulation at post-anthesis;NAA,nitrogen accumulation at pre-anthesis;NAPS,nitrogen accumulation per stem;NA,nitrogen accumulation;MSTN,maximum stem and tiller number.

5.Conclusion

Grain yield and NUE were synchronously increased among wheat cultivars released after 2000 in the Yangtze River Basin. The effective ears and single-spike yield jointly promoted the grain yield of the high-yield and-NUE cultivars. The more effective ears were achievedviaincreasing tiller fertility rather than the maximum stem and tiller number. The greater single-spike yield was explained by single-stem biomass at post-anthesis,resulting from higher leaf stay-green ability and flag leafPn. High-yield and -NUE cultivars had remarkable NUpE and accumulated more N in leaf,stem and leaf sheath,and unit leaf area. In addition,tiller fertility was crucial for improving grain yield and NUE.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31771711 and 32071953),the National Key Research and Development Program of China (2016YFD0300405),the Priority Academic Program Development of Jiangsu Higher Education Institutions,China,the Project of the Vice General Manager of Science and Technology of Jiangsu Province,China(FZ20211472),and the Plan of Gathering 1 000 Leading Talents of Suqian,China.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.10.005