How delaying post-silking senescence in lower leaves of maize plants increases carbon and nitrogen accumulation and grain yield

Rongfa Li,Dandan Hu,Hao Ren,Qinglong Yang,Shuting Dong,Jiwang Zhang,Bin Zhao,Peng Liu*

State Key Laboratory of Crop Biology/College of Agronomy,Shandong Agricultural University,Tai’an 271018,Shandong,China

Keywords:13C-Photosynthate distribution Nitrogen uptake Maize grain yield Delaying lower leaf senescence Post-silking

ABSTRACT Planting maize at high densities leads to early leaf senescence,and the resulting reduction in the number of lower leaves affects the plant’s root function and lowers its grain yield.However,the nature of the process by which lower leaf senescence affects biomass accumulation and grain yield formation in maize is not clear.This study aimed to shed light on how these factors are related by investigating the effects of the plant growth regulator 6-benzyladenine(6-BA)on the senescence of lower leaves of maize plants.In two maize cultivars planted at densities of 67,500(low density,LD)and 90,000(high density,HD)plants ha-1,plants treated with 6-BA maintained a high green leaf area index (LAI) longer than control (CK)plants,enabling them to maintain a higher photosynthetic rate for a longer period of time and produce more biomass before reaching physiological maturity.Spraying the lower leaves of maize plants with a 6-BA solution increased the distribution of 13C-photosynthates to their roots,lower leaves and bracts,a result that can be ascribed to a decreased retention of 13C-photosynthates in the stem and grain.In both seasons of the experiment,maize plants treated with 6-BA accumulated more N in grain and maintained a higher N content in roots and leaves,especially in lower leaves,than CK.Increased C assimilation in the lower leaves may explain why N uptake in plants subjected to the 6-BA treatment exceeded that in CK plants and why both photosynthesis rate and dry matter accumulation were maintained throughout grain filling.Our results suggest that a suitable distribution of C and N in leaves post-silking may maintain plant root function,increase N use efficiency,maximize the duration of high LAI,and increase grain yield.

1.Introduction

The world population is expected to increase to 8.5,9.7,and 11 billion by 2030,2050 and 2100,respectively[1].Current crop production levels cannot meet the food demands of such populations[2].Given the limited arable land area available for food production,the only way to meet future demand is to increase the crop yield per unit area of arable land [3].This goal is particularly important for maize,which,as China’s largest food crop,crop production accounting for about 40%of national[4,5],plays a key role in China’s food security [6,7].

Increasing maize planting density is an effective way to increase its grain yield per unit area[8,9].However,planting density affects the post-silking source–sink ratio by altering the leaf area,light interception,and kernel number of individual plants [9,10],causing a higher planting density to accelerate leaf senescence,in turn reducing the photosynthesis rate after silking[11].Leaf senescence can reduce yield by reducing LAI,shortening the time during which the leaf photosynthesis rate can be maintained at a high level,or regulating nutrient-transfer efficiency and harvest index [12–15].

Optimizing plant architecture and delaying leaf senescence may increase the interception of solar radiation and the production of photosynthate during the post-silking stage [16].The combined effect of delaying leaf senescence and maintaining the level of photosynthesis may produce more assimilates for growing grain,thus creating potential for maximizing the grain yield [17].In previous studies,application of benzyl adenine (BA) increased the grain weight of maize [5,18,19] and wheat [20,21].The highest yields were obtained after a relatively long growing season,with leaves remaining green until grain formation had reached the maturity stage [22].Delaying the harvest until after the maize grain has reached the maturity stage is an effective measure for increasing cumulative temperature utilization in China’s wheat–maize rotation systems [23,24].

Maintaining the green leaf area of maize plants may affect their root nutrient-absorption ability after silking,because roots require energy for nutrient absorption [25].Senescence in a leaf is regulated by its N content [26],and can be delayed by application of 6-BA,which prolongs its photosynthetic function and results in a higher grain yield[5].In addition,increased N absorption after silking may increase the longevity of a plant’s leaves,increasing postsilking biomass accumulation and yield formation[27].The growth of roots is highly dependent on the availability of photosynthates from the shoot,and as allocation of C to the root decreases [28],absorption of N by the root also decreases,resulting in an imbalance between C and N metabolic rates [29].The grain yield of maize depends on the supply of C and N,which are nutrient sources in the photosynthetic assimilates formed and transported to the kernels [12,30].

Previous studies[11,31,32]have investigated ways to delay leaf senescence and maintain root function.While agronomic measures could delay lower canopy senescence in high-density plant populations,there have been few reports on the effects of those measures on plant physiological processes,N absorption and distribution,post-silking biomass accumulation,and yield formation.The objectives of this study were to(i)assess the correlation between lower leaf senescence and biomass accumulation and grain yield;and(ii)measure and characterize the physiological effects of spraying 6-BA in terms of the delay in senescence of lower leaves of maize plants and the increase in maize yield,with respect to C and N accumulation and distribution in the plants.

2.Materials and methods

2.1.Experimental site

Experiments were performed at the experimental farm of the State Key Laboratory of Crop Biology and Shandong Agricultural University (36°10′N,117°9′E,151 m above sea level) during the 2016 and 2017 growing seasons.The 0–20 cm soil layer showed the following characteristics:available N content 0.73 g kg-1,rapidly available phosphorus (P) content 25.83 mg kg-1,rapidly available potassium(K)content 104.62 mg kg-1,and organic matter content 11.32 g kg-1.

2.2.Experimental design

The maize cultivars Denghai 661 (DH661) and Zhengdan 958(ZD958) were sown at planting densities of 67,500 (LD) and 90,000 (HD) plants ha-1.The experimental design was a randomized complete block design with four replications,and each plot was 120 m2in size (20 6 m,length width),accommodating 10 rows of maize plants spaced 60 cm apart.All plots were supplied with N,P2O5,and K2O at rates of 200,72,and 96 kg ha-1,respectively.The entire amount of P and K fertilizers and 50% of the N fertilizer were applied before sowing.The remaining 50%of the N fertilizer was applied at the V12 stage.After sowing,the plots were irrigated at 500 m3ha-1to ensure germination.On days 10,20 (R2),and 30 (R3) after silking,a 100 mg L-16-BA solution(Sigma,San Antonio,TX,USA)was applied to the lower leaves(defined as ranging from the base leaf to the second leaf below the ear leaf)using an electric sprayer with a single nozzle at a rate of 300 L ha-1.The 6-BA treatment was applied at dusk on a sunny day.The control (CK) population was treated with distilled water [19].The weather conditions at the experimental site in 2016 and 2017(including daily precipitation and daily minimum and maximum temperatures) are presented in Fig.1.

2.3.Sample collection

2.3.1.Measurement of LAI

The leaf area of six plants per plot was measured using a Portable Area Meter (LI-3000C,LI-COR,Lincoln,NE,USA).The leaves were divided into the following categories:upper-layer leaves(from the top to the second leaf above the ear leaf),middle-layer leaves (the three leaves representing the ear leaf and the first leaves above and below the ear leaf),and lower-layer leaves(from the second leaf below the ear leaf to the base leaf).The measurements were used to determine LAI,using the following formula:

2.3.2.Measurement of photosynthetic rate (Pn)

The leaf photosynthetic rates in leaves from five plants per plot were measured between 09:00 AM and 12:00 AM at the R1,R3,and R5 stages using a portable photosynthesis system (CIRAS-III,PP System,Hansatech,UK).The following leaves were sampled:the third leaf above the ear leaf,representing the upper leaf layer;the ear leaf,representing the middle leaf layer;and the third leaf below the ear leaf,representing the lower leaf layer.

2.3.3.Measurement of H2O2concentration

The amount of H2O2generation in leaves at the R3 stage was measured using the diaminobenzidine (DAB) staining method developed by Chen et al.[33].Leaves from plants subjected to the 6-BA treatment described in Section 2.2 were collected and stained using a 1 mg mL-1DAB solution with a pH of 3.8 at 37°C for 2 h.After DAB staining,the leaves were placed in boiling ethanol for 10 min and then cooled to room temperature and photographed.

To determine the H2O2concentration,0.05 g of leaf sample was homogenized in 2 mL of 0.1% trichloroacetic acid (TCA) solution.The homogenate was centrifuged at 12,000 g and 4 °C for 30 min.After centrifugation,0.5 mL of the supernatant was added to 1.5 mL of 10 mmol L-1potassium phosphate(pH 7.0),and 1 mL of 1 mol L-1KI solution.After incubation of the resulting leaf extract in darkness for 1 h,its H2O2content was calculated from its light absorbance at 390 nm using a standard curve prepared with multiple concentrations of H2O2[34].Each treatment was repeated six times.

2.3.4.Tagging of selected plants with13CO2

In 2017,ten representative plants from each plot were selected at the silking stage and radioactively tagged with13CO2isotopes to permit the measurement of C distribution in various parts of the plant (see also 2.3.7).To tag the plants,the ear leaves of each selected plant were wrapped in Mylar bags with a thickness of 0.1 mm,thus restricting the sunlight reaching the leaves to 95%of its natural intensity.After the bottoms of the bags were sealed with Silly Putty (M&G Chenguang Stationery Co.,Ltd.,Shanghai,China),they were filled with 60 mL of13CO2gas (99 atom %)between 9:00 and 11:00 AM.After photosynthesis was allowed to proceed for 60 min,the13CO2in each bag was extracted with KOH cleaner to absorb any remaining13CO2isotopes,after which the bags were removed [34].

2.3.5.Measurement of biomass

Fig.1.Minimum and maximum temperature and precipitation at the study site in 2016 and 2017.

At the R1 and R6 stages,five plants were sampled.Each plant was separated into the following parts:root,stalk (including bracts,tassels,leaf sheaths,and cobs),upper-layer leaves,middle-layer leaves,lower-layer leaves,and grain.The roots were excavated,retaining a soil volume of 60 25 60 cm(length width depth)for each plant.After excavation,the roots were separated from the soil by vigorous rinsing with water at low pressure and separated into embryonic and nodal roots.After measurement of root length,all sample components were oven-dried at 105 °C for 30 min and then dried at 80 °C to constant weight and finally weighed to obtain the biomass.

2.3.6.C and N accumulation and distribution

All samples from the plants radioactively tagged as described in Section 2.3.5 were ground using an MM 400 mixer mill (Retsch,Germany).The13CO2concentrations in each of the plant components described in Section 2.3.6 were determined using Isoprime 100 Analyzer(Isoprime,UK).The13CO2abundance in each component was calculated as follows:

where RPBD(C isotope ratio) was taken to be 0.0112372.The13CO2content in each component was calculated as follows:

The13CO2partitioning in each component,which is the amount of recovered isotope,expressed as a percentage of the total amount of isotope used for tagging the whole plant,was calculated as follows:

To measure N concentration,samples were ground into a fine powder,digested with 20 mL H2SO4–H2O2,and analyzed with an Auto Analyzer III (SEAL,Hamburg,Germany).N accumulation was calculated by multiplying the N concentration by the dry matter weight.

2.3.7.Grain yield and yield components

To determine grain yield (adjusted to 14% moisture content),the middle three rows (5 1.8 m,length width) in every plot were harvested,and 40 ears from each plot were used to determine 1000-kernel weight and kernel number per ear.The measurement was repeated for three replicates.

2.4.Statistical analysis

Statistical analysis of the measurements was performed with SPSS (SPSS Inc.,Chicago,IL,USA).The least significant difference(LSD) method was used to compare treatments at the level of P 0.05.LAI,H2O2production,Pn,13C and N content,and biomass were subjected to one-way ANOVA to identify significant differences among the treatments.

3.Results

3.1.Leaf senescence

Fig.2.Effect of 6-BA treatment on green leaf area at densities of 67,500 (LD) and 90,000 plants ha-1 (HD).Each symbol represents the mean ± SD of three replicates.

Fig.2 shows the LAI at various growth stages for each layer of the canopy.In both years,leaves from the lower leaf layer of ZD958 plants showed a lower LAI than those of DH661.The upper leaf layers,in contrast,showed the opposite trend at the R1 stage.Increasing the planting density increased the LAI.Plants sprayed with 6-BA showed higher LAI than plants from the CK population.

Increasing planting density increased LAI at the R1 and R3 stages,but promoted senescence in leaves from the lower layer.At the R5 stage,the LAI of leaves from the lower layer of DH661 plants from LD populations that were subjected to 6-BA treatment was 67.3% lower than the LAI measured at the R1 stage,and for plants from HD populations it was 76.8% lower.For ZD958 plants,the corresponding reductions in the LAI for plants from LD and HD populations were 63.7% and 77.1%,respectively.Compared with plants subjected to 6-BA treatments,the lower layer of leaves from plants subjected to the CK treatment featured a dramatically decreased LAI at the R5 stage (Fig.2).With respect to plants subjected to the CK treatment,the LAI of leaves from the lower leaf layer of DH661 plants from LD populations at the R5 stage was 73.2%lower than that at the R1 stage,and for plants from HD populations it was 85.0% lower.The corresponding reductions for leaves of ZD958 plants were 70.2% and 81.7%.

3.2.Leaf H2O2 content

Leaves from lower leaf layers produced more H2O2than leaves from middle and upper leaf layers.Higher planting densities increased leaf senescence and H2O2accumulation (Fig.3).When plant density was increased from LD to HD,the H2O2content in the upper,middle and lower leaf layers from DH661 plants was increased by 13.5%,23.8% and 11.5%,respectively,and the corresponding increases in leaves from ZD958 plants were 4.9%,21.0%,and 32.4% (Fig.3).

3.3.Photosynthetic rate

The photosynthetic rates in plants sprayed with 6-BA at different growth stages were significantly higher than those in plants from the CK populations.At the R5 stage,the photosynthetic rate in leaves from the upper layer of DH661 plants from LD populations treated with 6-BA was 24.0% higher than that in leaves from the CK population,and for HD populations the corresponding increase was 10.9%.The same measurements for leaves from the middle layer showed increases of 21.7% for LD and 30.5% for HD populations,and for leaves from the lower layer the corresponding increases were 60.4%and 51.5%.For ZD958 plants from LD and HD populations,the photosynthetic rates in leaves from the upper layer were respectively 8.5% and 7.1% higher than those in leaves from plants from the CK population,while the corresponding rate increases in leaves from the middle layer were 6.3%and 17.0%,and those in leaves from the lower layer were 19.2%and 15.3%(Fig.4).

3.4.Biomass accumulation and distribution

Leaves from both DH661 and ZD958 plants treated with 6-BA supported higher photosynthetic activity and remained green longer.The 6-BA treatment promoted dry matter accumulation during the post-silking stages (Fig.5).On average,the biomass accumulated during the post-silking stages in DH661 plants from LD populations subjected to 6-BA treatment was 18.3%higher than that accumulated in plants from HD populations,and the corresponding increase in plants from HD populations was 15.2%.For ZD958 plants,the increases in plants from LD and HD populations were 14.3% and 13.4%,respectively.With respect to the total biomass accumulated in DH661 plants,6-BA treatment achieved an increase of 8.7% over the CK population in LD populations and of 10.2% in HD populations.For ZD958 plants,the corresponding increases were 9.5% in plants from LD populations and 6.2% in plants from HD populations.

Fig.3.Effects of plant density on the senescence of leaves in three leaf positions and H2O2 elevation at the R3 stage in 2016.The upper panel shows qualitative DAB staining of H2O2.The bottom panel shows quantitative H2O2 determination.Each symbol represents the mean ± SD of three replicates.Values accompanied by different letters are different at P ＜0.05.

Fig.4.Effect of 6-BA treatment on net photosynthetic rate in leaves from the lower,middle and upper leaf layers at densities of LD and HD at the R1,R3,and R5 stages.The lower leaf layer is represented by the third leaf below the ear leaf,the middle leaf layer is represented by the ear leaf,and the upper leaf layer is represented by the third leaf above the ear leaf.LD,67,500 plants ha-1;HD,90,000 plants ha-1.Each symbol represents the mean ± SD of three replicates.

Fig.5.Effect of the 6-BA treatment on dry matter accumulation at densities of 67,500 (LD) and 90,000 plants ha-1 (HD).Each symbol represents the mean ± SD of three replicates.Values accompanied by different letters are different at P ＜0.05.

Spraying 6-BA affected the distribution of photosynthates across different plant organs at the R6 stage.At R6,the distribution of13C-photosynthates in the roots,lower leaves and bracts of plants sprayed with 6-BA had increased,but in stems and grain it had decreased.Compared to the CK population,spraying plants in LD populations with 6-BA increased the mean13Cphotosynthate content in roots,lower leaves,and bracts by 18.6%,23.7%,and 11.3%,respectively,and for plants from HD populations,the corresponding increases were 18.9%,38.8%,and 12.9%.For plants from LD populations subjected to 6-BA treatment,however,the13C-photosynthate content in stems was 7.3% lower than that in plants from the CK population,and in grain it was 1.2%lower.For plants from HD populations,the corresponding reductions were 4.6% and 2.1% (Table 1).

3.5.N accumulation and distribution

Total N accumulation at the R1 stage in plants from a given cultivar did not differ between the 6-BA and CK treatments (data not shown).In DH661 plants,the N content per plant at physiological maturity was higher than that in ZD958 plants.DH661 plants from LD populations subjected to the CK and 6-BA treatments showed mean N contents of 4.50 and 3.65 g per plant,respectively,and the corresponding values in plants from HD populations were 5.08 and 4.04 g per plant.ZD958 plants from LD populations subjected to the CK and 6-BA treatments showed mean N contents of 4.78 and 4.15 g per plant,respectively,and the corresponding values in plants from HD populations were 4.04 and 3.95 g per plant(Table 2).

Compared with the CK treatment,the 6-BA treatment achieved an increase in N content of 12.8%in plants from LD populations and an increase of 20.8% in plants from HD populations.In both seasons,maize plants treated with 6-BA also showed higher N accumulation in their kernels (3.8%–15.6%),while maintaining higher N contents in their roots (12.5%–60.0%) and leaves (10.9%–35.6%),especially in lower leaves (11.1%–76.9%).There were differences in N accumulation and distribution among densities and cultivars.For both LD and HD populations,the N content of the root,stem,kernels,and middle-layer leaves of DH661 plants at the maturity stage was higher than that for ZD958 plants (Tables 2;S1),and the N content of leaves from the middle and upper leaf layers was higher than that of leaves from the lower leaf layer.The relationship between N uptake by the shoot and root biomass at maturity indicates that a linear model is a good fit for the experimental data (P ＜0.01) (Fig.S1).

3.6.Grain yield and yield components

The maize cultivar,the planting density,and whether plants were sprayed with 6-BA all significantly affected kernel numberper ear,grain weight,and yield(Table 3).In HD populations,ZD958 plants showed a significantly higher grain yield than DH661 plants after 6-BA treatment (Table 3).Under other treatments,however,the yield from DH661 plants tended to be higher than that from ZD958 plants.

Table 1 Effect of 6-BA and CK treatments on 13C-photosynthate distribution(%)in maize plants of the DH661 and ZD958 cultivars at densities of 67,500(LD)and 90,000 plants ha-1(HD)in 2017.

Table 2 Effect of 6-BA treatment on N content at densities of 67,500 (LD) and 90,000 plants ha-1 (HD).

Table 3 Effects of 6-BA on grain yield and yield components at densities of 67,500 (LD) and 90,000 plants ha-1 (HD).

As expected,increasing planting density increased grain yield.Averaged over two years,the yield from HD populations of DH661 plants was 11.5% higher than that for LD populations of the same cultivar,and the corresponding increase for ZD958 plants was 18.2% (Table 3).Planting density did not affect the number of ears per hectare,with the exception of the number of ears achieved by the LD population of ZD958 plants in 2017.Spraying 6-BA significantly increased grain yield in both LD and HD populations,resulting in increases of respectively 9.1% and 8.4% over the CK populations.Higher grain yields were due mainly to increased kernel numbers per ear.For plants from LD populations subjected to 6-BA treatment,the number of kernels per ear was 3.1% higher than that for the corresponding CK populations,and for plants from HD populations it was 2.1% higher.For DH661 plants,the grain weight was higher than that for ZD958 plants,but no significant differences were found between the different treatments at the same planting density.With respect to components comprising grain yield,the number of ears per hectare and kernel number per ear contributed more to grain yield increase than did grain weight (Table 3).

3.7.How leaf senescence,N uptake and root weight relate to grain yield and post-silking biomass

Grain yield was positively associated with post-silking biomass and negatively associated with reduction in LAI,Pn,and root weight after silking(Table S3).Reductions in the LAI of leaves from the lower leaf layer were positively associated(r=0.625,P ＜0.01)with post-silking root weight reductions and reductions in the Pnof leaves from the upper leaf layer,and negatively associated(r=0.816,P ＜0.01) with post-silking biomass accumulation.

4.Discussion

4.1.Leaf senescence

Increasing planting density accelerated leaf senescence,particularly for leaves from the lower canopy layer (Figs.2 and 3).If the LAI falls below a certain critical value,the reduced availability of photosynthates reduces grain growth[11].Because increases in maize yield may be restricted by source activity[35,36],genotypes that exhibit delayed aging may have a capacity for prolonging the period during which they can maintain photosynthesis in the canopy,suggesting that they can be used to increase yield[36,37].

4.2.C and N accumulation and distribution

In our experiment,reductions in LAI occurred after the R3 stage(especially in the lower leaf layer of plants of the ZD958 cultivar),a phenomenon that is indicative of carbohydrate remobilization from the leaves to the ear and a smaller amount of carbohydrates being transported to the root [38–41].The amount of13Cphotosynthates in the roots,lower leaves,and bracts of plants sprayed with 6-BA was higher than that in plants from the CK population,but in shoots it was lower(Table 1).Limited carbohydrate supply and increased N transport in the root caused the root length and activity of maize grown in the field to decrease rapidly during the post-silking stages[28,39,42].In maize,the growth potential of new shoots drives N uptake and is usually associated with the root[28,43].Early leaf senescence may compromise the ability of the root system to absorb nutrients from the soil or cause an imbalance in the source–sink ratio in the latter growth season.At maturity,there was a positive correlation between root biomass and shoot N uptake at maturity (Fig.S1).

A higher level of post-silking N uptake may assist with leaf longevity,which increases post-silking biomass accumulation and thereby yield [27,32].The greenness of the leaves will affect the growth of the root system,thereby increasing the capacity of the root to absorb nutrients;in turn,a strong ability of the root system to absorb N delays leaf senescence (Fig.S1).6-BA affects not only the allocation of C between shoots and roots,but also the absolute amount of accumulated C.Post-silking and total N uptake by plants of both cultivars that were subjected to 6-BA treatment were higher than those for plants from the CK population(Table 2).

Delayed leaf senescence increased the source–sink ratio during grain filling and led to the allocation of a higher proportion of assimilates to the roots [26,36],findings echoed by ours (Table 1).Improving maize root parameters like root dry weight can increase N uptake and yield[9].In this study,DH661 plants subjected to 6-BA treatment in 2016 and 2017 accumulated more biomass than ZD958 plants,and this increase was accompanied by greater N accumulation at the R6 stage (especially at low plant densities).Also,canopy senescence of DH661 plants was delayed (Fig.4;Table 2).Increased biomass accumulation and N uptake during the post-silking stage by plants subjected to the 6-BA treatment may be associated with increased sink strength.The interaction between the planting density and the effects of the 6-BA treatment greatly in uences the ratio between total N content in aboveground plant components and the green leaf area (Fig.4;Tables S1,2),and this effect can be seen in the changes in photosynthesis and biomass (Figs.3 and 5).N content in the root,stem,upperlayer leaves,middle-layer leaves,lower-layer leaves,and kernels was positively correlated with yield (r=0.716–0.905) (P ＜0.01)(Table S2).

4.3.Yield and yield components and dry matter accumulation

Grain yield and kernel number are associated with the slower senescence rate of lower leaves [38,39].Delaying leaf senescence and prolonging the period during which photosynthesis can be maintained may stimulate kernel development,suggesting 6-BA treatment as a potential method for maximizing the grain yield from maize [17].In the present study,the yield from plants subjected to 6-BA treatment was higher than that from the CK population,and the yield increase could be ascribed mainly to the persistence of the capacity of leaves from the middle and lower leaf layers to maintain photosynthesis (Table 3;Fig.5).Application of 6-BA increased the number of kernels per ear,which is a yield component strongly associated with grain yield(Table 1).Delaying the loss of green leaf area and extending the time during which leaves have photosynthetic capacity may also increase the interception of light by the canopy [36].Delaying the senescence of leaves after silking increased dry matter accumulation(Figs.2 and 5),in agreement with Zhang et al.[38].The relative increase(10.5%–20.8%)in post-silking DM production was slightly higher than the relative increase (4.6%–14.4%) in grain yield.

4.4.N-use efficiency

The effects of regulatory measures on leaf senescence and grain yield have received little attention,especially from the angle of improving the N-use efficiency(NUE)of maize.In our study,plants subjected to 6-BA treatment achieved both higher biomass accumulation and greater total N content than plants from the CK population(Fig.5;Table 2),and showed differences in stem and leaf N content(Table 2).Plants subjected to the 6-BA treatment showing a higher NUE than plants from the CK population,but also a lower N harvest index (NHI) and internal NUE (NUtE) (Table S1).The retention of N in leaves may reduce the availability of N in grain,which would explain the decrease in NHI as a ‘‘stay-green cost”incurred upon the leaves.A reduced sink capacity hinders the transportation of carbohydrates,causing the stems and leaves to remain rich in soluble sugars,while a poor conduction capacity of the stem and sheath affects the absorption of N by the root system,which in turn causes a decrease in plant N accumulation and an increase in C and N content (Fig.S2).

5.Conclusions

Compared with plants from the CK population,plants that were sprayed with 6-BA maintained a higher photosynthetic rate in their leaves and exhibited increased levels of post-silking13Cphotosynthates in their roots,resulting in increased post-silking N-uptake,and biomass.Other major contributors to increased grain yield were greater post-silking biomass and N accumulation,both the result of increased levels of13C-photosynthates in roots.These positive effects also led to greater N accumulation in kernels,contributing to higher N-use efficiency.A suggestion for a future study is to consider the adequacy of C partitioning to roots and shoots at post-silking stages.

CRediT authorship contribution statement

Rongfa Li:Writing–original draft,Conceptualization,Writing–review &editing.Dandan Hu:Methodology,Data curation,Writing– review &editing.Hao Ren:Visualization,Investigation.Qinglong Yang:Visualization,Investigation.Shuting Dong:Methodology,Data curation,Resources.Jiwang Zhang:Methodology,Data curation.Bin Zhao:Methodology,Data curation.Peng Liu:Conceptualization,Visualization,Writing– review &editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Acknowledgments

This study was financially supported by the National Key Research and Development Program of China (2016YFD0300106,2018YFD0300603) and the Shandong Modern Agricultural Technology &Industry System (SDAIT-02-08).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.11.006.