Effects of nitrogen fertilizer and chemical regulation on spring maize lodging characteristics,grain filling and yield formation under high planting density in Heilongjiang Province,China

LIU Xiao-ming,GU Wan-rong,Ll Cong-feng,LI Jing,WEI Shi

1 College of Agronomy,Northeast Agricultural University,Harbin 150030,P.R.China

2 Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,P.R.China

Abstract Now,lodging is a major constraint factor contributing to yield loss of maize (Zea mays L.) under high planting density.Chemical regulation and nitrogen fertilizer could effectively coordinate the relationship between stem lodging and maize yield,which significantly reduce lodging and improve the grain yield. The purpose of this study was to explore the effects of chemical regulation and different nitrogen application rates on lodging characteristics,grain filling and yield of maize under high density. For this,we established a field study during 2017 and 2018 growing seasons,with three nitrogen levels of N100 (100 kg ha–1),N200 (200 kg ha–1) and N300 (300 kg ha–1) at high planting density (90 000 plants ha–1),and applied plant growth regulator (Yuhuangjin,the mixture of 3% DTA-6 and 27% ethephon) at the 7th leaf. The results showed that chemical control increased the activities of phenylalanine ammonia-lyase (PAL),tyrosine ammonia-lyase (TAL),4-coumarate:CoA ligase (4CL),and cinnamyl alcohol dehydrogenase (CAD),and increased the lignin,cellulose and hemicellulose contents at the bottom of the 3rd internode,which significantly reduced the lodging percentage. The lignin-related enzyme activities,lignin,cellulose and hemicellulose contents decreased with the increase of nitrogen fertilizer,which significantly increased the lodging percentage. The 200 kg ha–1 nitrogen application and chemical control increased the number,diameter,angle,volume,and dry weight of brace roots. The 200 kg ha–1 nitrogen application and chemical control significantly increased the activities of ADP-glucose pyrophosphorylase (AGPase),soluble starch synthase (SSS) and starch branching enzyme(SBE),which promoted the starch accumulation in grains. Additional,improved the maximum grain filling rate (Vmax) and mean grain filling rate (Vm),which promoted the grain filling process,significantly increased grain weight and grain number per ear,thus increased the final yield.

Keywords:lodging resistance,grain filling,starch synthesis,yield,maize

1.Introduction

High planting density is the predominant way to improve maize yield in recent years (Chenet al.2010; Tokatlidiset al.2011). In a certain range,increasing planting density can affect the canopy structure,improve the light use efficiency and improve the population productivity. However,at high densities,growth rate per plant decline and young kernel abortion are aggravated (Tollenaar and Aguilera 1992;Borráset al.2007). In addition,high planting density intensified competition among individuals for resources,lead to weak stems and inhibited root extension,thus increased lodging risk (Liuet al.2010).

Lodging is the permanent displacement of stems from an upright position to the vertical direction (Pinthus 1974;Berryet al.1999). Lodging was divided into root lodging and stem lodging according to the location and characteristics of lodging in maize (Becket al.1988). A lot of factors contribute to root lodging,such as the number of root,root diameter and growth direction (Hebert 1992; Stam 1992).Previous studies have reported that stem quality was the prominently indicator affecting stem lodging (Martin and Russell 1984). Cellulose,hemicellulose and lignin,as the main components of the stem,have a significant impact on the lodging resistance of the stem (Appenzelleret al.2004; Hussainet al.2020). The yield loss caused by stem lodging was more serious than that caused by root lodging.Stem lodging damaged the transport system and prevented water and nutrient transport between root and overground part,which led to a reduction in photosynthetic products transported from leaves to ear,and maize yield decreased(Becket al.1988). Lodging significantly restricted the yield and yield increasing potential. The effect of lodging on yield could be realized by affecting grain sink capacity and grain number (Berryet al.2004). In the filling period,lodging reduced the grain filling rate by 0.5% each day (Stapper and Fischer 1990). For every 1% increase in lodging rate,the yield decreases by approximately 108 kg ha–1(Sangoiet al.2002). Lodging has become one of the important factors that affect yield formation and grain formation and production efficiency in current crop systems.

The application of plant growth regulators is an effective way to reduce lodging. Chemical control can optimize plant morphology and increase yield by regulating plant endogenous hormone signaling and metabolism (Naeemet al.2012; Zenget al.2012). Previous studies have shown that chemical control play critical roles in improved the plant morphology and structure,increased stem bending resistance,thereby contributing to reduce lodging rate (Xuet al.2017). Chenet al.(2011) reported that chemical control improved the lignin content by improving the lignin related enzyme activities,which enhanced stem quality and lodging resistance. Shiet al.(2019) has reported that chemical control can promote brace root development,thereby reducing root lodging of maize. Furthermore,chemical control could enhance photosynthesis and photosynthate partitioning,significantly improve in grain development and yield (Waqaset al.2017). Yuhuangjin is a type of plant growth regulator which has been widely used in maize production in China. It is the combination of ethephon and DA-6 with the function of enhancing lodging resistance and increasing yield of maize (Zhang Qet al.2014).

Nitrogen fertilizer plays an important role on improving maize grain yield and quality. The application of nitrogen enhanced the assimilate accumulation in the kernels after silking,and stimulated kernel setting and yield improvement(Ninget al.2018; Zhaoet al.2019). Starch is the main component of maize grain,contributes approximately 70%of the dry weight in grain. Therefore,the grain weight actually depend on the extent of starch accumulation in grain. Starch synthesis in grains is limited by the activity of starch synthesis enzyme (Jenner 1991). In fact,ADPglucose pyrophosphorylase (AGPase),granule-bound starch synthase (GBSS),soluble starch synthase (SSS),and starch branching enzyme (SBE) are several key enzymes of starch synthesis,which play an important role in stimulating starch accumulation and grain weight (Ball and Morell 2003; Tianet al.2009). Zhanget al.(2005) reported that sufficient nitrogen fertilizer supplying significantly increased the activities of AGPase,SSS and GBSS during grain filling,which promoted the synthesis and accumulation of starch,and profited to improve the grain filling process.

Heilongjiang Province is the major maize production base in China (Chai and Zhu 2016). In recent years,maize yield per unit area in Heilongjiang Province has been improved by increasing planting density and nitrogen fertilizer application.However,there are frequent lodging occurrences due to high planting density and unreasonable nitrogen fertilizer application in production,resulting in a significant reduction in maize yield (Shiet al.2016; Liuet al.2017). Lodging is one of the main factors restricting the further increase of maize yield in the cold region of Heilongjiang Province.Therefore,it is an important issue that must be solved to coordinate the relationship between lodging and yield.

We hypothesized that reasonable application of nitrogen fertilizer and chemical control can reduce the lodging risk and promote the yield formation under high planting density.To prove this hypothesis,this experiment measured the stalk physicochemical characteristics,root morphological features,grain filling characteristics,starch accumulation,key enzyme activity of starch synthesis and yield with different nitrogen fertilizer levels,and chemical control under high density. This study aims to provide a theoretical basis for increasing lodging resistance and maize yield under high density in Heilongjiang Province.

2.Materials and methods

2.1.Site description

The experiment was conducted in 2017 and 2018 at the experimental station of Northeast Agricultural University,Harbin,Heilongjiang Province,China (126°54´E,45°46´N,128.0 m a.s.l.). The experimental site soil was chernozem.The contents of total nitrogen,available phosphorus,available potassium,alkali-hydrolysable nitrogen,and organic matter of the upper 0–20 cm soil layer were 1.70 g kg–1,65.34 mg kg–1,179.35 mg kg–1,118.21 mg kg–1,and 25.25 g kg–1,respectively. The region is characterized as a temperate semi-humid continental monsoon climate,where the annual mean temperature and annual precipitation level are 4.5°C and 569 mm,respectively. Temperature and precipitation of the 2017 and 2018 experimental seasons are shown in Fig.1.

2.2.Experiment design and field management

The experiment was laid out as a split plot design with three replicates. The main plots were separately spraying Yuhuangjin (Y) or water (control),and the subplots were nitrogen fertilizer with three levels:100 kg ha–1(N100),200 kg ha–1(N200) and 300 kg ha–1(N300). The experimental chemical control was Yuhuangjin (the mixture of 3% DTA-6 and 27% ethephon,Haolun Co.,Ltd.,Fujian,China).Yuhuangjin was applied evenly on the leaf surface with spray applicator at a rate of 450 L ha–1at the 7th leaf stage,and the control sprayed water. The dose of Yuhuangjin was recommended from the manufacturer(0.83 mL L–1). Spring maize Longyu 365,a high-yielding variety in local production area,was sown manually at the density of 90 000 plants ha–1on 30 April while harvesting on 25 September for both years. The plots were 8 m long,with eight rows spaced 0.65 m apart. All plots were supplied with 100 kg ha–1P2O5and 100 kg ha–1K2O. Phosphorus and potassium fertilizer and half of nitrogen fertilizer (urea,46% N) were applied at the sowing stage. The other half of nitrogen was applied at the jointing stage. No irrigation water was applied during the maize growing season. Other management practices,such as pest control and tilling practices,were similar to conventional practices for planting maize.

2.3.Sampling and measurements

Stem chemical composition and lodging percentageTen plants were randomly selected in each treatment in central rows by avoiding boarder rows at elongation,tasseling,early filling,milk,and maturing stages. The third basal internode samples were dried at 80°C to a constant weight,crushed and sieved (0.25 mm) for determination. We investigated lodging plant number (N1) and total plant number (N) of maize in each experiment plot,and then calculated the lodging percentage (N1/N×100%). Additionally,stem lodging is stem breaking at or below the ear; and root lodging is stem bending over 45° from vertical.

The contents of cellulose,hemicellulose and lignin were determined by van Soestet al.(1991) abstersion fibre. First,0.5 g of dried samples were analyzed with acid detergent fiber (ADF) solution (20 g cetyl trimethylammonium bromide to 1 L of 1.00 N H2SO4),and determined ADF(cellulose+lignin) value.Second,samples were analyzed with neutral detergent fiber (NDF) solution (sodium lauryl sulfate,ethylendiamine-tetraacetic disodium salt dihydrate,sodium tetraborate decahydrate,sodium phosphate dibasic,anhydrous and triethylene glycol),and determined NDF(cellulose,hemicellulose+lignin) value. Finally,samples were analyzed with acid detergent lignin solution (72%H2SO4) and determined lignin value of samples. Cellulose content was obtained by calculating the difference between ADF and lignin contents,and hemicellulose content was obtained by calculating the difference between NDF and ADF contents.

Fig.1 Mean temperature (°C) and daily precipitation (mm) during 2017 and 2018 growing seasons.

PAL and TAL activities were determined by Wang Cet al.(2014). A total of 0.5 g of the sample was ground in a mortar on ice in 6 mL of sodium borate boric acid buffer (0.05 mol L–1,pH 8.8,containing 5 mmol L–1mercaptoethanol,1 mol L–1EDTA,a little PVP). The lapping liquid was centrifuged at 10 000 r min–1for 15 min at 4°C,and the supernatant was selected to determine the activity of PAL at the absorbance of 290 nm,and the activity of TAL at the absorbance of 315 nm was determined.

4CL activity was referred to the method of Knobloch and Hahlbrock (1975). A total of 5 g of the sample was ground in a mortar on ice in 10 mL of 0.05 mol L–1Tris HCl buffer(pH 8.8,containing 0.014 mol L–1mercaptoethanol and 30%glycerin) with a little PVP and quartz sand. The lapping liquid was centrifuged at 10 000 r min–1for 15 min at 4°C,and the supernatant was selected to determine the activity of 4CL.

CAD activity was determined according to Morrisonet al.(1994). A total of 0.4 g of the sample was ground in a mortar on ice in 3 mL of extractionl buffer (0.1 mmol L–1phosphoric acid buffer,containing 15 mmol L–1mercaptoethanol,1 mmol L–1EDTA,pH 6.25) with a little PVP. The lapping liquid was centrifuged at 10 000 r min–1for 20 min at 4°C,and the supernatant was selected to determine the activity of CAD.

Morphological index of brace rootIn tasseling stage,three uniform plants were selected in each plot,and roots were digged by means of soil profile method. The root system in 0–30 cm soil layer was dug center on the plant with 1/2 plant spacing as the width and 1/2 row spacing as the length. The moisture on the surface was absorbed by tissue paper after rinsing the root,and then determined the root layers number by counting. The number,diameter,angle,and volume of the root were scanned and analyzed by a root scanner (WinRHIZO root system). The fresh brace roots were dried at 80°C to a constant weight and the dry weight was measured.

Grain filling characteristicsFive ears were selected that tasseled on the same day from each plot and the ears were sampled at 5-day intervals after silking. Hundred grains in the middle part of the ear were dried at 80°C to a constant weight and weighted separately. The grain filling process was fitted using a logistic growth equation as described by Zhang Let al.(2017):y=A/(1+Be–Ct). The days of reaching maximum grain filling rate wasTmax=(lnB)/C,the grain weight of reaching maximum grain filling rate wasWmax=A/2,the maximum grain filling rate wasVmax=(C×Wmax)/2,active grain filling period wasP=6/C,the effective filling period wast=(lnB+4.59512)/C,and the mean grain filling rate wasVm=A/t,respectively. Whereywas the grain weight (mg),twas the time after silking (d),Awas final grain weight (mg),andBandCwere coefficients determined by regression.

Starch content and starch synthesis key enzyme activityRepresentative ears were selected in maturing stage to determine starch content and starch synthesis key enzyme activity. Starch amylose and amylopectin contents were determined according to the “double-wave-length”method of He (1985). The main wavelength and a particular wavelength for amylase content determination were 620 and 479 nm,respectively; and those for amylopectin content determination were 556 and 737 nm,respectively. Total starch content was the sum of amylose and amylopectin contents.

AGPase,SSS and GBSS activities were determined according to Zhanget al.(2013). Ten grains were milled into flour in low temperature,and 5 mL extracting solution(100 mmol L–1Tricine-NaOH,8 mmol L–1pH 7.5 MgCl2,2 mmol L–1EDTA,12.5% (v/v) glycerol,1% (w/v) PVP-40,50 mmol L–12-mercaptoethanol) was added,then extracting solution was centrifuged 30 min at the speed of 1 000 r min–1in 4°C,supernatant was selected to determine the activities of AGPase and SSS; precipitation was washed twice by extracting solution,then was put into 2-mL extracting solution in suspension systems to determine the activity of GBSS.

SBE activity was determined according to Zhanget al.(2013). Ten grains were milled into flour in low temperature,5 mL citrate buffer (pH 7.0) was added,then extracting solution was centrifuged 20 min at the speed of 23 000 r min–1in 4°C,and supernatant was selected to determine the activity of SBE.

Yield and ear traitsAt maturing stage,9 m2of maize (three rows×5 m length per row) were harvested from the center of each plot to determine yield (moisture content was 14%).Twenty representative ears were selected in each plot to measure grain number per ear and 1 000-grain weight.Meanwhile,ear traits including length,width,row number per ear,kernel number per row and bald length were determined.

2.4.Statistical analysis

The data were summarized to calculate the mean value and standard error (SE) in two years. The mean values were compared by analysis of variance (ANOVA) to analyze the significant differences between samples with different nitrogen application rates and chemical control under high density (P<0.05). All statistical analyses were performed by SPSS 19.0 procedures (SPSS Inc.,Chicago,IL,USA).Microsoft Excel 2007 was used to draw figures.

3.Results

3.1.Stem physicochemical characteristics

Lignin,cellulose and hemicellulose are main components of cell wall,as well as indicators used for measuring the lodging resistance of maize stem. Nitrogen fertilizer and chemical control had significant effects on lignin,cellulose and hemicellulose contents in the 3rd basal internode under high planting density in 2017 and 2018. Lignin,cellulose and hemicellulose contents all rose and then fell from elongation to maturing stages,and the maximum value appeared at early filling stage (Fig.2). The lignin,cellulose and hemicellulose contents decreased with the increase of nitrogen fertilizer. Compared with N100 and N200,the lignin,cellulose and hemicellulose contents of N300 were lower by 5.5,7.8,7.4% and 1.9,4.4,3.9% in 2017 and lower by 5.9,6.4,7.5% and 3.0,2.7,3.1% in 2018 (mean of the data of five growth stages),respectively. Compared with the water treatment,chemical control increased the lignin,cellulose and hemicellulose contents by 11.4,13.7 and 11.3% in 2017,and 8.4,16.1 and 12.3% in 2018 (mean of the data of five growth stages).

Lodging occurred in different degrees at maturing stage under high planting density in 2017 and 2018. The lodging percentage increased with the increase of nitrogen fertilizer.Compared with N100 and N200,the lodging percentage of N300 increased by 60.0 and 48.1% in 2017 and 46.9 and 33.3% in 2018. Chemical control decreased the lodging percentage by 87.5 and 107.0% in 2017 and 2018. It indicated that applying chemical control and reducing the application of nitrogen fertilizer could decrease the risk of lodging under high planting density (Fig.3).

Correlation analysis demonstrated that the lodging percentage of maize was negatively correlated with the contents of lignin,cellulose and hemicellulose in the third basal internode of stem (Table 1). It indicated that increasing the contents of lignin,cellulose and hemicellulose in the 3rd basal internode of stem could reduce the lodging percentage,and lead to the improvement of the lodging resistance of maize stem.

The PAL activity of stem decreased during the growth period,specifically decreased rapidly in the early filling stage and then decreased slowly. The PAL activity decreased with the increase of nitrogen fertilizer,and chemical control significantly increased the PAL activity in 2017 and 2018(Fig.4-A and B).

The TAL activity rose and then fell from elongation stage to maturing stages in two years,which the maximum value appeared at the tasseling stage and remained stable from early filling stage. The TAL activity decreased with the increase of nitrogen fertilizer.Compared with water treatment,chemical control increased the TAL activity by 16.8 and 22.9% in 2017 and 2018 (Fig.4-C and D).

The CAD activity rose and then fell during maize growth stage in 2017 and 2018,and the maximum value appeared at the early filling stage. The CAD activity decreased with the increase of nitrogen fertilizer,yet the chemical control significantly increased the CAD activity (Fig.4-E and F).

The 4CL activity decreased during the growth period in 2017 and 2018,and increased slightly at milk stage. The 4CL activity decreased with the increase of nitrogen fertilizer,yet the chemical control significantly increased the 4CL activity (Fig.4-G and H).

Correlation analysis demonstrated that lignin content was positively correlated with the activities of PAL,TAL,CAD,and 4CL in the 3rd basal internode of stem (Table 2). It indicated that the increase in PAL,TAL,CAD,and 4CL activities was the enzymology basis for the increase in lignin content,thus improved the lodging resistance of stem.

3.2.Morphological characteristics of brace root

Nitrogen fertilizer and chemical control had significantly effects on morphological characteristics of brace root at the tasseling stage. With the increase of nitrogen fertilizer,the number,diameter,angle,volume,and dry weight of brace roots increased and then decreased,and the maximum values appeared in N200 treatment. Chemical control increased the number,diameter,angle,volume,and dry weight of brace roots by 19.4,10.5,28.3,15.7,and 17.6% in 2017 and 16.4,10.1,29.2,13.9,and 19.1% in 2018. Compared to N100+CK treatment,N200+Y treated plants had significant higher number of brace root plies in 2017,however,there was no significant difference in 2018(Table 3).

3.3.Grain filling characteristics

In the different nitrogen fertilizer and chemical control treatments,maize grain weight had a slow-fast-slow trend as “S” growth curve in 2017 and 2018 (Fig.5-A and B).The grain weight was increased by increasing nitrogen application,however,excessive nitrogen fertilizer inhibited the grain weight. The grain weight of N200 was significantly higher than that of N100 and N300,and chemical control significantly increased the grain weight. The grain filling rate showed a single peak curve during grain filling period,and the peak value appeared from 25 to 30 days after silking.With the increase of nitrogen fertilizer,the grain filling rate increased and then decreased,and the maximum value appeared in N200. Chemical control increased the grain filling rate in 2017 and 2018 (Fig.5-C and D).

Nitrogen fertilizer and chemical control had significant effects on the parameters of grain filling characteristics.With the increase of nitrogen fertilizer,Vmax,Vmand P increased and then decreased,and the maximum values of them appeared at N200 in 2017 and 2018. There were no significant effect onTmaxwith nitrogen fertilizer treatment in 2017 and chemical control treatment in 2018. Chemical control decreasedTmaxby 3.4–4.6%,and increasedVmaxandVmby 4.4–6.1% and 6.3–7.9% in 2017 and 2018. Chemical control reduced P in 2017,however,had no effect in 2018(Table 4).

Fig.2 Effect of chemical control and nitrogen fertilizer on the lignin,cellulose and hemicelluloses of stem under high density in 2017 (A,C and E) and 2018 (B,D and F). N100+CK,N200+CK and N300+CK,spraying water with the nitrogen application of 100,200 and 300 kg ha–1,respectively; N100+Y,N200+Y and N300+Y,spraying chemical control with the nitrogen application of 100,200 and 300 kg ha–1,respectively. Bars indicate standard errors of the means of three measurements. Different letters within a growth stage indicate significant difference at P<0.05.

3.4.Starch content and starch synthesis key enzyme activity

Nitrogen fertilizer and chemical control affected the starch synthesis and accumulation in grains during grain filling,and had a similar effect on starch content in two years.Total starch content increased and then decreased by increasing nitrogen application. The maximum value appeared at N200,which increased by 4.4–7.3% and 4.7–5.1% compared with N100 and N300 in two years.Nitrogen treatments had different effects on amylose and amylopectin contents. With the increase of nitrogen fertilizer,the amylase content in grain decreased,and the amylopectin content increased first and then decreased.Chemical control increased the amylopectin,amylase and total starch contents by 9.6–10.5%,3.8–5.2% and 7.7–8.8%in 2017 and 2018 (Table 5).

Fig.3 Effect of chemical control and nitrogen fertilizer on lodging percentage under high density in 2017 and 2018.N100+CK,N200+CK and N300+CK,spraying water with the nitrogen application of 100,200 and 300 kg ha–1,respectively;N100+Y,N200+Y and N300+Y,spraying chemical control with the nitrogen application of 100,200 and 300 kg ha–1,respectively. Bars indicate standard errors of the means of three measurements. Different letters within a growth stage indicate significant differences at P<0.05.

The activities of AGPase,SSS,GBSS,and SBE showed a single peak curve during grain filling process. The peak values of SSS,GBSS and SBE appeared at 20 d after silking and AGPase appeared at 30 d after silking. With the increase of nitrogen fertilizer,the activities of AGPase,SSS and SBE increased first and then decreased. The maximum value appeared at N200,which increased by 10.0–11.5%,9.7–10.8%,8.8–10.6% and by 5.7–7.5%,6.6–6.9%,4.4–6.3% compared with N100 and N300 in 2017 and 2018. The GBSS activity decreased with the increasing nitrogen application. Chemical control increased the activities of AGPase,SSS,GBSS,and SBE by 13.9–15.0%,13.2–13.8%,10.6–10.7%,and 11.3–14.02% in 2017 and 2018 (Fig.6).

3.5.Yield and ear traits

With the increase of nitrogen fertilizer,the grain number per ear,1 000-grain weight,and yield all increased and then decreased,and the maximum value appeared at N200. Chemical control significantly increased yield and its components. The highest yield was achieved in N200+Y treatment. The yield in 2017 was higher than that in 2018 under all treatments (Table 6).

With the increase of nitrogen fertilizer,ear width and rows number per ear increased and then decreased,and grains number per row increased in 2017 and 2018. The bald length reached the minimum value at N200. Chemical control significantly increased rows number per ear and grains number per row by 5.6–8.6% and 4.7–8.8%,and reduced bald length by 21.2–44.1% in 2017 and 2018 (Table 7).

3.6.Correlation analysis of starch content with starch synthesis key enzyme and yield

Correlation analysis demonstrated that amylopectin and total starch contents were positively correlated with the activities of AGPase,SSS and SBE,and amylose content was positively correlated with the activities of GBSS.Amylopectin and total starch contents were positively correlated with grain weight and yield (Table 8).

Table 1 Correlation coefficients of lodging percentage with lignin,cellulose and hemicellulose contents in the third basal internode1)

Fig.4 Effect of chemical control and nitrogen fertilizer on lignin synthesis-related enzymes (phenylalanine ammonia-lyase (PAL),tyrosine ammonia-lyase (TAL),cinnamyl alcohol dehydrogenase (CAD),and 4-coumarate:CoA ligase (4CL)) activities under high density in 2017 (A,C,E,and G) and 2018 (B,D,F,and H). N100+CK,N200+CK and N300+CK,spraying water with the nitrogen application of 100,200 and 300 kg ha–1,respectively; N100+Y,N200+Y and N300+Y,spraying chemical control with the nitrogen application of 100,200 and 300 kg ha–1,respectively. Bars indicate standard errors of the means of three measurements. Different letters within a growth stage indicate significant differences at P<0.05.

Table 2 Correlation coefficients of lignin content with lignin synthesis-related enzymes activities1)

Table 3 Effect of chemical control and nitrogen fertilizer on morphological trait of brace root under high density in 2017 and 2018

4.Discussion

4.1.Effect of nitrogen fertilizer and chemical control on lodging resistance

This study showed that the contents of lignin,cellulose and hemicellulose in the 3rd internode at the base of stem were significantly negative correlated with the lodging percentage of maize. It indicated that the increase in lignin,cellulose and hemicellulose contents in the basal internode would lead to reduce the lodging percentage and increase the lodging resistance of maize. The lignin content was significantly positive correlated with the activities of PAL,TAL,CAD,and 4CL,which was consistent with previous studies (Luet al.2014; Zouet al.2015). Previous study suggested that the improvement in the lodging resistance was primarily attributed to higher proportions of structural carbohydrates(e.g.,cellulose and lignin) (Zhang Jet al.2014). In this study,lignin,cellulose,and hemicellulose in the third basal internodes exhibited a single peak curve with the highest peak at early filling stage. The increase in structural carbohydrates from elongation to early filling stages is due to plant growth (Begovicet al.2015). However,the contents of lignin,cellulose and hemicellulose in the stem were reduced from early filling to maturing stages. It may be caused by carbohydrates translocated from the stem to the grain on account of the increase of the demand for nutrients in the grain after grain filling (Lemoineet al.2013; Longaresiet al.2019). In this study,as the increase of nitrogen fertilizer,the contents of lignin,cellulose and hemicellulose decreased,which decreased the basal internode filling degree of the stem and increased the lodging risk (Zhang M Wet al.2017). However,chemical control significantly increased the activities of lignin,cellulose,hemicellulose and the activities of lignin synthesis related enzyme,and effectively strengthened the basal internode the mechanical strength and reduced the lodging percentage at high planting density.

Brace root is an important part of maize root system,which plays an important role in absorbing water and nutrients during the later growth stage of maize. The morphology of brace root has significant effect on the growth,yield formation and lodging resistance of maize. Root lodging had some relationships with the number,diameter and growth direction of roots (Hebert 1992; Stam 1992). In addition,the increase of dry weight,length and absorption area of maize root available for coordinate the production,accumulation and transport of photosynthetic substances among the root,stem,leaf,and grain,accordingly improve the grain yield (Jinget al.2012; Qiet al.2014; Zhang Yet al.2014). In this study,with the increase of nitrogen application,the number,diameter,angle,volume,and dry weight of brace roots increased and then decreased. Chemical control significantly increased morphological characteristics of brace root. Our results suggest that 200 kg ha–1nitrogen application and chemical control treatment effectively promoted the growth of brace root under high density,which available for the increase of lodging resistance and coordination the functional balance between root system and overground part,and provided guarantee for the increase in grain yield.

Fig.5 Effect of chemical control and nitrogen fertilizer on grain weight and grain filling rate in 2017 (A and C) and 2018 (B and D). N100+CK,N200+CK and N300+CK,spraying water with the nitrogen application of 100,200 and 300 kg ha–1,respectively;N100+Y,N200+Y and N300+Y,spraying chemical control with the nitrogen application of 100,200 and 300 kg ha–1,respectively.Bars indicate standard errors of the means of three measurements.

Table 4 Effect of chemical control and nitrogen fertilizer on parameters of grain filling characteristics of maize in 2017 and 20181)

4.2.Effect of nitrogen fertilizer and chemical control on grain filling and starch synthesis characteristics

Grain filling is an important biological process of maize growth and development,which influences the final grain weight and yield. The effect of grain filling process on grain weight and yield is mainly determined by the grain filling rate and grain filling period (Johnson and Tanner 1972). Liuet al.(2013) believed that the grain weight was positively correlated with the mean grain filling rate and the maximum grain filling rate,but had less correlation with the active grain filling period. Our results suggest that 200 kg ha–1nitrogen application and chemical control treatment shortened theTmax,increased theVmandVmax,thereby leading to increased grain weight.

Starch is the main component of maize kernel,approximately accounting for 70% of the dry weight.Therefore,the grain filling process is mainly the process of starch synthesis and accumulation in maize kernel.Maize starch is composed of amylose and amylopectin,and the proportion and quantity of them in kernel affect the starch quality (Doehlertet al.1991; Steven and Takashi 1992; Morellet al.1995). In this study,with the increase of nitrogen application,the contents of amylopectin and total starch increased and then decreased,and the content of amylose decreased. Chemical control increased the contents of amylopectin,amylose and total starch. Starch is produced by the catalysis with a series of enzymes after the photosynthetic products are transported to grains in the form of sucrose. AGPase is mainly related to the total starch synthesis rate and the final starch content (Preiss 1991). SSS and SBE are mainly related to the synthesis of amylopectin,and ultimately affect the composition and structure of starch (Mizunoet al.1992; Fontaineet al.1993).GBSS mainly controls the synthesis of amylose and is the key enzyme to influence the ratio of amylose to total starch in grains (Martin and Smith 1995). Our study suggested that with the increase of nitrogen application,the activities of AGPase,SSS and SBE increased and then decreased,and the activity of GBSS decreased. Accordingly,the contents of amylopectin and total starch increased and then decreased,and the content of amylose decreased. Chemical control significantly increased the activities of AGPase,SSS,GBSS and SBE during grain filling,which promoted starch accumulation and increased starch content. The 200 kg ha–1nitrogen application and chemical control treatment promoted the synthesis and accumulation of starch in grains,which played an important role in grain filling.

4.3.Effect of nitrogen fertilizer and chemical control on yield

Nitrogen fertilizer and chemical control were effective measures to maintain a high level of yield (Wang Zet al.2014; Zhang Qet al.2014). In this study,200 kg ha–1nitrogen application and chemical control treatment not only reduced bald length and promoted ears growth,but also increased the two important yield components,grain number and 1 000-grain weight,and ultimately ensured to attain high yield. We considered that,200 kg ha–1nitrogen application and chemical control treatment increased maize yield at high planting density due to reduction of lodging by enhancing the content of structural carbohydrates in stem and promoting the growth of brace root. The improvement in stalk strength and brace root morphological characteristics contributed to increase the conduction of water,nutrients and photosynthate in plant,accordingly had a positive effect on grain filling process (Jinget al.2012; Gaoet al.2017;Xuet al.2017). The nitrogen fertilizer and chemical control stimulated grain filling process by improving starch synthesis related enzyme activities and promoting starch synthesis and accumulation in maize kernel,thus improved eartraits and significantly increased the yield. Hence,200 kg ha–1nitrogen application and chemical control improved lodging resistance and grain filling process of maize,which increased grain yield by increasing grain number and 1 000-grain weight. We conclude that 200 kg ha–1nitrogen application and chemical control might prevent maize lodging and increase grain yield under high planting density in Heilongjiang Province.

Table 5 Effect of chemical control and nitrogen fertilizer on the starch content of maize in 2017 and 2018

Fig.6 Effect of chemical control and nitrogen fertilizer on activities of ADP-glucose pyrophosphorylase (AGPase),soluble starch synthase (SSS),granule-bound starch synthase (GBSS),and starch branching enzyme (SBE) in maize grains in 2017 (A,C,E,and G) and 2018 (B,D,F and H). N100+CK,N200+CK and N300+CK,spraying water with the nitrogen application of 100,200 and 300 kg ha–1,respectively; N100+Y,N200+Y and N300+Y,spraying chemical control with the nitrogen application of 100,200 and 300 kg ha–1,respectively. Bars indicate standard errors of the means of three measurements. Different letters within a growth stage indicate significant differences at P<0.05.

Table 6 Effect of chemical control and nitrogen fertilizer on yield and yield components in 2017 and 2018

Table 7 Effect of chemical control and nitrogen fertilizer on ear traits of maize in 2017 and 2018

Table 8 Correlation analysis of starch content with AGPase,SSS,GBSS,SBE,and yield1)

5.Conclusion

In high planting density,200 kg ha–1nitrogen application and chemical control significantly increased the activities of PAL,TAL,4CL,and CAD,thereby increasing the contents of lignin,cellulose and hemicellulose,and improved the morphology of brace root lodging resistance. At the same time,it promoted the activities of starch synthesis key enzymes and the starch accumulation,and made the grain filling sufficient,ultimately increased the grain yield.

Acknowledgements

This study was supported by the National Key R&D Program of China (2016YFD0300103 and 2017YFD0300506),the Heilongjiang Provincial Funding for National Key R&D Programs of China (GX18B029) and the “Academic Backbone” Project of Northeast Agricultural University,China (17XG23).