Yield photosynthesis and leaf anatomy of maize in inter-and mono-cropping systems at varying plant densities

Hongwei Yng ,Qing Chi, *,Wen Yin ,Flong Hu ,Anzhen Qin ,Zhilong Fn ,Aizhong Yu,Ci Zho,Hong Fn

a State Key Laboratory of Arid Land Crop Science,Lanzhou 730070,Gansu,China

b College of Agronomy,Gansu Agricultural University,Lanzhou 730070,Gansu,China

c Institute of Farmland Irrigation,Chinese Academy of Agricultural Sciences/Key Laboratory of Crop Water Use and Regulation,Ministry of Agriculture and Rural Affairs,Xinxiang 453002,Henan,China

Keywords:Maize/pea intercropping Plant density Leaf area Photosynthesis Leaf anatomy

ABSTRACT Increasing plant density can increase cereal crop yields.However,the physiological and anatomical mechanisms of grain yield increase at high plant densities in maize-based intercropping systems are not well understood.A two-year field experiment was conducted in 2018 and 2019 to investigate grain yield,photosynthetic characteristics,stomatal traits,and leaf anatomy of maize plants in an intercropping system with high plant densities.Two cropping patterns (monocropping and intercropping) and three plant densities (D1,78,000 plants ha-1;D2,103,500 plants ha-1;D3,129,000 plants ha-1) were arranged in a randomized block design.Increasing maize plant density significantly increased maize yield,and intercropping gave a significant yield advantage over monocropping under the same plant density.Intercropping combined with high plant density increased the leaf area and SPAD value of maize,increasing the photosynthesis rates after the harvest of pea.At the twelfth leaf stage,the stomatal density and stomatal area of intercrops combined with medium plant density increased by respectively 10.5%and 18.4% relative to their values for the corresponding density of monocrops.Although leaf thickness of maize was reduced by increasing plant density,the chloroplast number and grana lamella number were higher in intercropping than in monocropping under different plant densities.These positive changes in leaf anatomy resulted in increased photosynthesis,suggesting a physiological basis for the increase in grain yield.

1.Introduction

Crop production must be increased by 70%–100% to satisfy the ever-increasing global demand for food,feed,and bioenergy[1,2].Appropriate crop management measure is a vital step to guarantee the global food security [3].Intercropping cultivation systems are effective measures to promote crop production through better use of farmlands and limited resources [4,5].Intercropping offers advantages over monocropping owing to temporal and spatial interspecific complementarity,making intercropping more tolerant to high plant density [6–8].Previous studies [7,9]have focused mainly on increases in grain yield,water use efficiency (WUE),and nitrogen use efficiency (NUE) in intercropping with high plant densities.Little attention has been paid to intercrops’ physiological and anatomical responses to high plant densities.

Photosynthesis is the primary determinant of crop yield.The efficiency with which crops capture sunlight and convert carbon dioxide and water into plant biomass is the key to final yield [9–11].The leaf is the main photosynthetic organ,and photosynthetic capacity can be increased by increasing leaf area [12].High planting density accelerated leaf senescence,reducing net photosynthesis rate after silking and assimilate availability for kernel formation[13,14].The stomatal opening,stomatal pore size,stomatal frequency (stomatal density and stomata index) and stomatal distribution pattern on leaf surfaces were changed under highdensity conditions,weakening gas exchange between the atmosphere and the plants and finally reducing grain yield [15,16].However,in another study [17],the leaf area and quality of main crops increased rapidly after short-growing crops were harvested in a strip intercropping system.These results indicate that intercropping increased crop productivity mainly via the recovery effect during the reproductive growth stage [18].It remains unclear whether this advantage is maintained under increased planting densities.

The anatomical structure of leaves can intuitively reflect the adaptability of plants to external environmental conditions[19,20].In the transverse direction,the morphology and internal structure of mesophyll cells influence photosynthetic capacity,and total mesophyll cell area per transverse section width (S/W)was associated with leaf area expansion[11,21].Chloroplasts were most sensitive to illumination and to morphological and structural changes associated with varying environmental conditions [22].Ren et al.[13]reported that chloroplast morphology and functional leaf ultrastructure were more damaged under high-than under conventional-density planting conditions.This damage is the main cause of reduced photosynthesis [23,24].It is thus desirable to investigate the effect of plant density on characteristics of leaf photosynthesis at the cellular and sub-cellular level in intercropping systems.

Maize/pea strip intercropping is a promising model for increased crop-production sustainability in northwestern China.In this system,maize expressed a high potential for greater yield owing to differentiation in spatial resources:leaves and shoots for light and roots for water,in contrast to maize monocropping[25,26].Pea,as a short-growing crop,is harvested at the early tasseling stage of maize.We hypothesized that intercropping has a higher density tolerance because it maintains good photosynthetic physiological characteristics with high yield compared to monocropping.These hypotheses were investigated by 1)measuring the effect of maize plant density on grain yield under monocropping versus intercropping production patterns,and 2)determining whether plant density and intercropping with pea favorably alters the photosynthetic capacity and anatomical characteristics of maize,leading to higher yield.

2.Materials and methods

2.1.Site description

A field experiment was conducted during the growing seasons(April to September) of 2018 and 2019 at the Oasis Agricultural Research Experiment Station of Gansu Agricultural University(37°96′N,102°64′E.1776 m above sea level),located in Wuwei,Gansu province,China.The field location is in a typical arid region in the cold temperate zone.The long-term (1960–2016) annual mean temperature was 7.2°C with cumulative temperatures above≥0 and 10°C of 3646°C and 2985°C.Average frost-free period was 156 days.Average annual sunshine duration was ＞2945 h,and total annual solar radiation was 6000 MJ m-2.The region is suitable for the intercropping production pattern.The field is classified as a desert soil filled with calcareous particles,with 0.78 g kg-1total nitrogen (N),1.14 g kg-1available phosphorus (Olsen phosphorus),14.3 g kg-1soil organic matter,and pH 8.2.During the growth seasons of 2018 and 2019,total precipitation was 273.1 and 171.3 mm,and average temperature was 18.1 °C and 17.9 °C,respectively.Climate data (Fig.1) were obtained from the Wuwei Meteorological Bureau.

2.2.Experimental design

The experiment design was a randomized complete block design with two factors.The first factor was cropping pattern,with two treatments:intercropping and monocropping,and the second factor was maize plant density,with three treatments:low density(78,000 plants ha-1),medium density (103,500 plants ha-1),and high density(129,000 plants ha-1).Different maize plant densities were formed by keeping row spacing constant while changing plant-to-plant distance (Fig.2).In intercropping plots,pea and maize were planted in 190-cm wide strips,with 8/19 occupied by pea (a strip 80 cm wide,with 20-cm row spacing),and the remaining 11/19 occupied by maize (a strip 110 cm wide,with 40-cm row spacing).The intercropped species received the same planting densities used for monocropping,based on the occupied area of each component.The densities of intercropped maize were 45,000,60,000,and 75,000 plants ha-1.The seeding rate of intercropped pea was 760,000 plants ha-1.As a conventional tillage practice,the field was plowed to 30 cm depth after maize harvest,and in the next spring,maize strips were covered with 0.01 mm plastic film after fertilizing,harrowing,smoothing,and compacting.

Maize (cv.Xianyu 335) was sown on April 19,2018,and April 21,2019,and pea (cv.Longwan 1,a popular needle-leaf cultivar)was sown on March 28,2018 and April 1,2019.Maize was harvested on September 28,2018 and September 26,2019,while intercropped pea was harvested earlier on July 10,2018 and July 8,2019.Straw was removed from field after harvest.Urea (46-0-0 of N-P2O5-K2O) and diammonium phosphate (18-46-0 of N-P2O5-K2O) were evenly broadcast and incorporated into the 0–30 cm soil layer by shallow rotary tillage prior to seeding.Sole pea received 136 kg N ha-1and 67.5 kg P2O5ha-1;sole maize received 360 kg N ha-1and 180 kg P2O5ha-1.Intercrops received the same rates of fertilization on an area basis as sole crops.Pea received all N and P as basal fertilizer.Maize received 30% of N and all P as basal fertilizer at sowing,50% at jointing,and 20% at grain-filling.For the last two N applications for maize,a 3-cm diameter hole (10 cm deep) was made 5 cm from the maize stem,fertilizer was applied into the hole,and the hole was sealed with soil.Drip irrigation was used for the entire experimental area.All plots received 120 mm water in the previous fall just before soil freezing,and then five successive irrigations of 75,90,90,75,and 75 mm water were applied at the jointing,pre-heading,silking,flowering,and filling stages of maize,while 75 and 90 mm,respectively,were applied at pea branching and flowering stages for sole pea plots.In each intercropping plot,both pea and maize received the same area-based irrigation quota as the corresponding sole cropping treatment.

2.3.Measurements and calculation

2.3.1.Grain yield and yield components

Maize grain yields were determined by harvesting all plants at maturity in a sampling area of 7.7 m2(7-m length of three rows)at the center of each plot.The harvested kernels were air-dried,cleaned,and weighed.Grain moisture content was measured with a SYS-PM-8188 grain moisture meter (Saiyasi Co.,Ltd,Liaoning,China).The grain moisture measurements were calculated at 14%moisture content as the standard for maize storage and sale in China[27].To determine yield components including the ear characteristics,number of kernels per ear,and 1000-kernel weight,10 plants were randomly selected from yield-measured plots.

2.3.2.Leaf area

Ten representative plant samples were marked in each plot at the sixth leaf stage (V6),twelfth leaf stage (V12) and blister stage(R2) to be later used for measurement of leaf length (L) and maximum leaf width(W).The following equation was used to calculate leaf area [28]:

where 0.75 is the compensation coefficient of the maize leaf.

Fig.1.Precipitation and air temperature during the growing seasons(from March 1 to October 1)of maize–pea intercropping in 2018 and 2019 at Wuwei experiment station,Gansu province,China.

Fig.2.System layout of maize/pea intercropping at the Oasis Experimental Station of Gansu Agricultural University in Wuwei,Gansu province,China in 2018 and 2019.(a)Monocropped maize at jointing stage.(b)Intercropped maize at jointing stage.(c)Three maize planting densities:low density,78,000 plants ha-1;medium density,103,500 plants ha-1;high density,129,000 plants ha-1)in a monocropping system.(d)Three maize planting densities in an intercropping system.The planting density of intercropped maize was 11/19 that of monocropping maize.

2.3.3.Leaf gas exchange and fluorescence

Leaf gas exchange measurements were performed on tagged leaves using an LI-6800 portable open infrared gas analysis system(LI-COR Inc.,Lincoln,NE,USA) equipped with a leaf chamber fluorometer (6800-01A;LI-COR).Measurements were performed on sunny days at 9:00–11:00 AM to avoid stomatal closure during the middle of the day.Photosynthesis was measured in middle parts of maize leaves at V6,V9,V12,R1,R2 and R5 stages as advised by Ritchie et al.[29].Prior to the tasseling (VT) stage[29],the youngest fully expanded leaf was used for measurement.From then on,the ear leaves were measured.For the intercropping system,the pea sides maize leaves were selected to determine photosynthetic characteristics from three readings per leaf.On each measurement date,three leaves from each sub-block were adapted to dark for 15 min.A value of 1200 μmol m-2s-1(90%red and 10% blue light) of photosynthetic photon flux density was set to match,as closely as possible,the natural light wavelength.The CO2concentration in the leaf chamber was set to 400 μmol mol-1and the leaf temperature was maintained at 25°C with relative humidity of 55%and a flow rate of 500 μmol s-1.After acclimation to steady state,the photosynthesis rate was measured for approximately 10 min.Net photosynthetic rate(Pn),minimal fluorescence (Fo),and maximal fluorescence (Fm) were recorded.Optimal/maximal photochemical efficiency of PS II in the dark was calculated based on the following equation proposed in [30–32]:

2.3.4.Relative content of chlorophyll (SPAD)

After measurement of photosynthesis,the same leaf position was selected to measure chlorophyll relative content with a SPAD chlorophyll meter (SPAD 502,Minolta Camera Co.,Ltd.,Tokyo,Japan) using three readings.Measurements avoided major veins and were made in the field between 9:00 and 11:00 AM.

2.3.5.Scanning and transmission electron microscopy

Three stages (V6,V12,and R2) of maize leaf growth were selected for scanning and transmission electron microscopy (SEM and TEM) just after measurement of photosynthesis.For SEM,leaf sections (1–3 mm2) were collected from the middle parts,which matched gas exchange assessment positions (one leaf position per plant from three plants with four replications per treatment).Each sample was then placed in an Eppendorf tube,fixed with 2.5% glutaraldehyde (Solarbio Co.,Ltd.,Beijing,China) for at least 4 h at 4 °C.Leaf materials were then washed three times with phosphate buffer (pH 7.4) for 30 min and then fixed with 1%osmium tetroxide for 4 h at 4°C,followed by dehydration through an ethanol gradient series of 10%-100%.The samples were coated with gold,and electron micrographs were obtained with the Hitachi Science System SEM(S-3000N,Hitachi,Tokyo,Japan).The pretreatment of TEM was the same as for SEM samples.Cells were collected as described above and the samples were embedded in Epon 81.semi-thin (1 μm) and ultrathin (90 nm).Cross-sections were prepared with an LKB-5 ultramicrotome (LKB Co.Ltd,Uppsala,Sweden).The semi-thin cross-sections were stained with toluidine blue and examined with a Nikon Eclipse E600 microscope(D7000,Nikon Co.,Ltd,Kyoto,Japan) equipped with a Nikon 5 MP digital microscope camera (DS-Fi1;Nikon Co.,Tokyo,Japan).Images were captured at 200× magnification.Ultrathin sections were examined with the transmission electron microscope H-7650;(Hitach) after contrasting with 2.0% uranyl acetate (w/v)and lead citrate.Chloroplast cell images were obtained at 3000–30,000 magnifications.Anatomical data were extracted from the images using Image J software (National Institutes of Health,Bethesda,MD,USA).According to the Hu et al.mothed [11],Measurement illustration for structural parameters is presented in Fig.S1.

Stomatal traits were examined using STM images with ≥30 fields of examination for each treatment.In each microscopic field(0.129 mm2),stomatal density (SD),pavement cells (PC),area of stomatal pore(SA),and stomatal length(SL)and width(SW) were measured.Stomatal and pavement cells were counted in five quadrats on each leaf side and their densities were expressed as mean number per mm2.The following equation was used to calculate stomatal index [33]:

Mesophyll cell traits and leaf thickness were measured using semi-thin cross-section images obtained from ≥ 30 fields of examination per treatment.In each field,the mesophyll cell range was framed to determine cell density and cell area per transverse section width (S/W) and total mesophyll cell length of cells exposed to intercellular air space.This measurement was performed avoiding the vascular bundle.Chloroplast traits included chloroplast thylakoid membrane organization and number of thylakoids per granum,assessed and documented as electron micrographs.

2.4.Statistical analysis

Data were analyzed with SPSS17.0 (SPSS Inc.,Chicago,IL,USA).Analysis of variance (ANOVA) was fitted for all dependent variables.Means were compared by ANOVA followed by Duncan’s multiple range test.

3.Results

3.1.Grain yield and component

During the two experimental years,increasing plant density increased the grain yield of both the inter-and the monocropping systems (Table 1).In the monocropping system,maize yields at medium and high plant densities were greater than those at low plant density by respectively 8.8% and 5.3%,and in the intercropping system,the differences were 12.8% and 6.3%,respectively,indicating that intercropping had a higher tolerance of high plant density.Overall,number of ears (×104m-2),number of kernels per ear,and 1000-kernel weight of maize in intercropping were increased by respectively 5.3%,4.8%,and 3.7% relative to monocropping (Table 1).Increasing maize plant density increased the number of ears,but reduced the number of kernels per ear and 1000-kernel weight.Number of kernels per ear at medium plant density was significantly reduced by 2.7%–5.6% relative to low density under monocropping,but was not significantly different under intercropping.The 1000-kernel weight at low,medium and high densities were 3.4%,2.3% and 5.6% higher under intercropping than under the corresponding monocropping in both years.The study indicated that increasing the number of ears and maintaining a higher number of kernels per ear and 1000-kernel weight under higher plant density explained why intercropping had the advantage of tolerating density.

3.2.Leaf area and chlorophyll content

Increasing maize planting density increased leaf area in intercropping at reproductive stage of maize (Table 2).At the V6 stage,average of maize leaf area was reduced by 24.8% with intercropping compared to the monocropping.Increasing the maize plant density,leaf area of medium and high maize plant density was 16.5% and 32.5% less than that of low plant density (Table 2).However,the maize leaf areas were respectively 13.7% and 9.2%higher in intercropping than in monocropping at the V12 and R2 stages of maize (after pea harvest).Additionally,the ID2 treatment continuously showed the highest values for leaf area.On average,the leaf area of maize with ID2 was increased by 24.3%and 14.3% at the V12 stage and by 7.9% and 9.9% at R2 stage,compared to SD1 and SD2,respectively.Low,medium and high density with intercropping significantly increased SPAD by 12.0%,15.8% and 6.8% after V12 stage,compared to that with corresponding densities of monocropping (Fig.3).These results indicated that maize leaf area and SPAD were increased after pea harvest,providing the basis for greater yield under intercropping with high plant density.

Table 1 Yield components and yield of maize as affected by cropping pattern and plant density.

Table 2 Leaf area per plant as affected by cropping pattern and plant density.

3.3.Photosynthesis and stomatal characteristics

To identify the effects of plant density on maize growth and development in intercropping systems,it was necessary to examine gas exchange processes attributed to photosynthesis and stomatal traits.Increasing plant density increased maize photosynthesis(Pn)after pea harvest in the intercropping system(Fig.4).Averaged across each year,ID2 generated the highest Pn,which was increased by 19.0% and 9.1% compared to that of SD1 and SD2,respectively.The Fv/Fmof maize leaves under ID2 was increased by 14.6% and 20.1% relative to SD1 and SD2 at the R5 stage.However,ID3 and SD3 reduced Fv/Fmby 12.6% and 28.2%,respectively,compared to SD1,implying that high plant density inhibited the photosynthetic capability of the maize leaf but less severely under intercropping than under monocropping.

Fig.3.The SPAD value of maize leaves at low(a),medium(b)and high maize density(c)in intercropping and monocropping and the mean values of whole growth stages(e,f)during 2018 and 2019.Treatment codes represent 78,000 plants ha-1,103,500 plants ha-1,and 129,000 plants ha-1 of maize plant density in monocropping(SD1,SD2,and SD3)and intercropping(ID1,ID2,and ID3),respectively.V6,sixth leaf stage;V9,ninth leaf stage;V12,twelfth leaf stage;R1,silking stage;R2,blister stage;R5,dent stage.The planting density of intercropped maize was 11/19 that of monocropping maize.Bars in(a–c)are LSD of net photosynthetic rate at each sampling period.Different letters in(e)and (f) indicate significant difference at P ＜0.05 among treatments.

During the whole growth stage,stomatal length (SL),stomatal width(SW),stomatal density(SD),stomatal area(SA)and stomatal index (SI) were significantly higher under the intercropping than under monocropping (Table 3;Figs.S2,S3).Increasing plant density increased all of these indicators.However,high maize plant density significantly reduced SL,SW,and SC of maize leaf by 7.6%,11.2%,and 17.6%,respectively,compared to low maize plant density.At V6 stage,stomatal traits showed almost the same values for all treatments,but lower SW,SC,and SA were observed under monocropping with high maize plant density (SD3).The SD,SA,and SI of maize leaves were greater at the V12 than at the V6 stage,especially in intercropping,and SD and SI were increased by 15.4%,23.4%,and 26.2% under ID2 relative to SD1.The variations in SL,SW,SC and SA in response to different treatments were consistent with that at V6 stage.The stomatal trait parameters were decreased at R5 stage,but compared with monocropping,intercropping had a higher SA and SI at the different densities.Thus,the intercropping system maintained more favorable gas exchange conditions than did the monocropping system at all tested plant densities.

3.4.Leaf anatomical characteristics

At the V6 stage,marked differences were observed in leaf thickness(LT),chloroplast number per mesophyll(ChlN),plastid globule numbers per chloroplast (PG),and grana number per chloroplast(G) between intercropping and monocropping systems (Table 3;Figs.S4,S5).The bundle sheath cell area (BSCA)was much greater under intercropping than under monocropping.ChlNand ChlWdecreased with increasing plant density in given field areas.PG and G increased with plant density under monocropping but decreased under intercropping (Table 4;Fig.S4).

At V12 stage,LT decreased with increasing plant density,but this adverse effect was alleviated in intercropping (Table 4).The effect on mesophyll thickness (MT) in the monocropping pattern was consistent with that at V6 stage but increased under intercropping.The S/W and BSCA were 14.3 and 8.8% lower under ID2 than SD1.No significant difference in ChlWwas detected among density levels under intercropping and monocropping.However,G,ChlNand ChlLwere 8.2%,16.7%,and 27.1% higher under ID2 than SD1,owing probably to the maintenance of elongated spindle shape of chloroplast with increasing plant density (Table 4;Fig.S5).At R5 stage,differences in LT and MT among density levels were larger under intercropping than under monocropping.

3.5.Correlation

Pearson correlations were calculated between grain yield,leaf area,SPAD,and photosynthesis.At V6 stage,leaf area and net photosynthetic rate were significantly and negatively correlated with maize grain yield (Table S1).A positive correlation (P ＜0.01) was found between maize grain yield and leaf area at the V12 and R2 stages.A positive correlation (P ＜0.05) was found between grain yield and stomatal density (SD) at the V12 stage.Thus,increased leaf area and stomatal density were key factors ensuring better grain yield in the late growth stage of maize.Correlation was also observed between leaf area and anatomical traits at the three tested growth stages (Table S2).LT,MT,S/W,and ChlNat the V12 and R2 stages were positively correlated with maize leaf area,but negatively correlated at the V6 growth stage.In contrast,only bundle sheath cell area(BSCA)at the V12 stage was negatively correlated with leaf area.Compared with the V6 stage,grana number per chloroplast (G) was positively correlated (P ＜0.05) with leaf area at V12 and R2 stages (Table S1),indicating that increased numbers per chloroplast(ChlN)and grana numbers per chloroplast(G) can be considered indicative of an increased photosynthesis capability of the leaf.

Table 3 Leaf stomatal features of maize leaves at sixth stage (V6),twelfth (V12),and blistering stage (R2) as affected by plant density in 2019.

Fig.4.The net photosynthetic rate(Pn)of maize leaves with low(a),medium(b)and high maize density(c)in intercropping and monocropping and the mean value of whole growth stages(e,f)during 2018 and 2019.Treatment codes represent 78,000,103,500,and 129,000 plants ha-1of maize plant density in monocropping(SD1,SD2,and SD3)and intercropping (ID1,ID2,and ID3),respectively.V6,sixth leaf stage;V9,ninth leaf stage;V12,twelfth leaf stage;R1,silking stage;R2,blister stage;R5,dent stage.The planting density of intercropped maize was 11/19 that of monocropping maize.Bars in(a–c)are LSD of net photosynthetic rate at each sampling period.Different letters(e)and (f) indicate significant difference at P ＜0.05 among treatments.

4.Discussion

4.1.Impacts of intercropping and plant density on maize production

Under proper water and fertilizer management,increasing plant density is one of the keys to increasing crop yields [34].However,excessive planting densities may impair the light structure of crop groups,causing low light conditions.This limits plant growth owing to a shortage of ATP supplied by photosynthesis [35,36].Excessive plant density negatively affected crop development and reproductive functions.Previous study [37] has confirmed that excessive plant density reduced grain yield.Similar results were found in our study,especially for monocropping.Adoption of high populations accelerated interplant competition for light,water and nutrients,inducing apical dominance and barrenness and ultimately reducing the number of ears per plant and kernels set per ear [38].Intercropping is an agronomic practice widely used in many countries,including China [3,25,32].In northwest China,maize/pea intercropping is a popular agronomic practice that has been shown [39] to increase grain yield relative to monocropping.

In the present study,grain yield of intercropping was greater under the different plant densities than that of monocropping under the corresponding plant densities (Table 1).Some researchers[40,41]have attributed the yield advantage of intercropping to the weaker marginal superiority of maize(the weaker the marginal superiority,the higher the grain yield).In another study [39],the positive effect of intercropping promoted higher root density and greater leaf area,driving maize to use more soil water and solar radiation in intercropped pea strips.One study[42]suggested that maize can withstand high plant density through a sort of selfdefense mechanism,by which its leaf area size is adjusted to respond to the unfavorable environment of high density.Similarly,the present study showed reduced leaf area with increasing maize plant density before harvest of pea (V6 stage),with a smaller leaf area under intercropping than under monocropping(Table 2).This finding may be attributed to the competitive advantage of the pea crop during the maize–pea symbiosis period,delaying the growth and development of maize in comparison with sole maize [5].Increased maize planting density resulted in further intensification of the competition between maize and pea [26].However,at the V12 stage/after pea harvest,intercropping increased the leaf area of maize in comparison with monocropping (Table 2).

The advantage of intercropping has been attributed [8,43] in part to the unique phenomenon of ‘‘restorative growth”.Phenotypic plasticity allowed intercropped maize to take advantage of resources (light,space,nutrients) that became available after the dominant pea was harvested,leading to more rapid nutrient acquisition[4,7]and accelerating the elongation rate of the leaf[9].This process eventually led [43] to 20%–70% greater maize biomass accumulation under intercropping than under monoculture.The positive effect was also shown by the increase in leaf area,which provided additional photosynthetic potential (Figs.3,4),and assisted the recovery and growth of later crops [9,12].Stomata on the leaf surface that controlled gas exchange between the atmosphere and plants also influence potential plant growth and crop performance [15].Plant leaves optimized gas exchange efficiency by changing their stomatal density and index.In our study,the stomatal length of ID2 had greater than those of the other treatments (Table 3).The stomatal width decreased with increasing plant density,while stomatal density,area,and index increased under ID2 treatment after pea harvest (Table 3;Figs.S2,S3).Stomatal density and net photosynthetic rate were positively correlated (Table S2).Better stomatal traits further increased net photosynthetic rate [11],and also considered another plausible reason for the higher photosynthetic efficiency of the leaves in later growth stages under intercropping at different plant densities[13,19].Fv/Fmis an indicator of leaf photosynthetic efficiency[44].In our study,intercropped maize leaves maintained higher Fv/Fmvalues than those under monocropping (Fig.5),further promoting higher photosynthetic rates under intercropping than under monocropping during the reproductive growth of maize.

4.2.Changes of leaf anatomic structure with cropping pattern and planting density

We used an anatomical approach to explore the microstructure of intercropped maize leaves at different growth stages.In previous studies [20,22],leaf area was affected by cell size and number.Those researchers used the alteration in S/W to explain the mechanism of leaf area expansion in winter oilseed rape,showing that S/W was negatively correlated with leaf expansion[11].In our study,though increasing maize plant density reduced S/W,no difference was observed between monocropping and intercropping (Table 4).Thicker leaves offer an advantage in photosynthesis because they have a larger mesophyll surface area per unit leaf area,which is filled with chloroplasts to secure an area for CO2dissolution and transport [12].This might explain the higher photosynthetic capacity at the medium plant density along with intercropping.However,leaf thickness decreased at high plant density,most likely owing to decreased mesophyll cell size under high density that restricted the increase in leaf thickness[45].This finding was supported by our study in the form of a strong correlation between leaf thickness and mesophyll thickness (Table S2).Previous study [46] also found that unfavorable environment led to more and smaller vascular bundle sheaths(BSCA) in plant leaves,a response that might aid C4 plant leaves to adapt to their environment and increase their carbon assimilation efficiency.In our study,the BSCA of maize was negatively correlated with planting density,and intercropping increased the values over those under monocropping,especially at the R2 stage (Table S2).

Fig.5.The Fv/Fm of maize leaves in intercropping and monocropping at six stages during 2018 and 2019.Treatment codes represent 78,000 plants ha-1,103,500 plants ha-1,and 129,000 plants ha-1of maize plant density in monocropping (SD1,SD2,and SD3) and intercropping (ID1,ID2,and ID3),respectively.V6,sixth leaf stage;V9,ninth leaf stage;V12,twelfth leaf stage;R1,silking stage;R2,blister stage;R5,dent stage.The planting density of intercropped maize was 11/19 that of monocropping maize.Bars are LSD of net photosynthetic rate at each sampling period.

Fig.6.A schematic diagram showing the physiological and anatomical mechanisms of increased maize yield and plant density tolerance under intercropping.

The number,size,and internal structure of chloroplasts,the main photosynthetic organelles,strongly influence photosynthetic performance[22,47].Our results showed a marked decrease in the number of chloroplasts and grana with increasing maize plant density (Table 4;Fig.S4).The observed decreases in ChlNand grana explained the decrease of photosynthetic pigments and net photosynthetic rate under monocropping [48].In other words,the high photosynthetic rate of ID2 treatment was due mainly to increases in ChlNand grana over the corresponding values under monocropping,especially in the late growth period.These results explain why higher grain yield was obtained under intercropping at several maize densities than under monocropping.Under high maize plant density,the number of grana decreased,owing to underdevelopment or disintegration of some grana.Some chloroplasts swelled and became round,and the thylakoid structure swelled and was arranged in a disorderly pattern,while the intracellular part of the membrane was destroyed and also induced apoptosis[49,50].This was most likely the main reason for the decline in photosynthesis.

From a psychoanalytical viewpoint, the changes that appear in the 1857 version reveal a great deal about Wilhelm {Grimm}. To begin with, the betrayal of the father can be equated81 with Wilhelm s father s early death. The mistreatment of the girl and her helpless condition can be connected to the mistreatment Wilhelm endured in Kassel, his asthma82 and heart troubles. The creation of the Strong angelic figure who helps the girl can be related to Jacob {Grimm}, who constantly stood by Wilhelm and came to his aid. The misunderstandings in the marriage that are patched up by the angel may indicate some difficulties in Wilhelm s marriage with Dortchen Wild that were resolved by Jacob. Finally, the general theme of the story can be summed up by the Grimms family motto: Tute si recte vixeris - he cannot go wrong whose life is in the right. (Brothers, 171)

It can be concluded that high maize plant density impairs maize performance,especially under monocropping.Integration with intercropping and high plant density changed leaf area size,leading to the modification of leaf anatomy.These changes in both leaf area size and leaf anatomy increased photosynthetic capacity and thereby maize grain yield (Fig.6).

5.Conclusions

We suggest that high density tolerance under intercropping is one of the major reasons for higher grain yield than under monoculture.In the maize/pea intercropping system,increasing maize plant density increased leaf area,stomatal density,leaf thickness,and number of grana per chloroplast after an intercropped pea harvest.The positive changes in both leaf area and leaf anatomy increased photosynthetic capacity and thereby maize grain yield.Our results contribute to better understanding the physiological mechanisms by which the intercropping production pattern shows great potential for increasing maize yield in arid and semi-arid regions and worldwide.

CRediT authorship contribution statement

Hongwei Yang:Data curation,Writing– original draft,Investigation.Qiang Chai:Conceptualization,Writing–review&editing,Funding acquisition,Supervision.Wen Yin,Falong Hu and Anzhen Qin:Writing– review &editing.Aizhong Yu and Cai Zhao:Project administration.Zhilong Fan and Hong Fan:Investigation.

Declaration of competing interest

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

Acknowledgments

This work was supported by the Central Government Guide Local Science and Technology Development Project (ZCYD-2021-10),the China Agriculture Research System (CARS-22-G-12),the Science and Technology Program of Gansu Province (20JR5RA037,20JR5RA025,and 20JR5RA008),and the National Natural Science Foundation of China (31771738,32101857).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.09.010.