Influence of plastic film mulching and planting density on yield, leaf anatomy, and root characteristics of maize on the Loess Plateau

Li Niu, Ynyn Yn, Peng Hou, Weno Bi, Rulng Zho, Yonghong Wng,Shokun Li, Tisheng Du, Ming Zho, Jiqing Song,, Wenin Zhou,

aKey Laboratory of Prevention and Control of Residual Pollution in Agricultural Film, Ministry of Agriculture and Rural Affairs, Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100081, China

bCollege of Water Resources and Civil Engineering, China Agricultural University, Beijing 100083, China

cInstitute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

dNingxia Academy of Agriculture and Forestry Sciences, Crops Research Institute, Yongning 750105, Ningxia, China

ABSTRACT

1. Introduction

Climate change reduces the productivity of major agricultural crops around the world[1,2].With continued global warming,changes in the hydrological cycle are expected to increase the incidence of drought [3,4]. Agriculture needs to adapt quickly to the changing climate to ensure more efficient use of water,while maintaining food security for the fast-growing world population. Appropriate water and crop management practices are needed to achieve these goals [5-8]. The Loess Plateau in northwestern China has a typical arid and semiarid climate, with an annual average rainfall of 250-600 mm and predominantly dryland agriculture [9]. In addition to low spring temperatures in this region, water shortage and high evaporation, due to inadequate and unpredictable rainfall,and inadequate solar radiation are the major factors limiting crop production[10].

Maize is one of the major crops on the Loess Plateau,accounting for 27.3% of the total agricultural area [11]. In recent years, plastic film mulching has been widely used for maize cultivation in this region, as it greatly reduces soil evaporation, increases soil moisture, and increases topsoil temperature [12-15]. It also improves the soil surface microclimate by increasing sunlight reflectance and resistance to air flow [16] and reduces weed and pest pressure [17].

The application of plastic film mulching for crop management in arid and semi-arid regions has been studied extensively. Previous studies have focused mainly on the quantification of the growth, development, yield, quality and water use efficiency of crop plants. These studies [18-24]showed that mulching increases crop yield and quality and increases water use efficiency by promoting soil moisture and heat preservation. At the physiological level, crops grown under plastic film mulching exhibit higher chlorophyll content [21], RuBisCO activity [25], antioxidant activity [26],electron transport rate [27] and heat dissipation [21] than crops grown without mulching. Thus, plastic film mulching increases the photosynthetic capacity of crop plants.

The anatomical structure of leaves plays an important role in the adaptation of plants to environmental conditions, and changes in leaf anatomy affect leaf photosynthesis [28-30].Engineering crop leaf structure could be used to increase the photosynthetic efficiency of crop plants and consequently crop yield [31]. Although leaf anatomy is recognized as a major factor affecting leaf photosynthesis,very little attention has been paid to the adaptation of leaf anatomy to crop management practices, and the mechanisms underlying the effect of leaf anatomy on photosynthesis under plastic film mulching conditions remain unknown.

The ability of plants to absorb water and minerals from the soil is attributed primarily to the extensive root system,which determines the impact of agricultural practices on soil, shoot function, and crop yield. Plastic film mulching enhances root growth as well as water and nutrient uptake[32-34];however,few studies have investigated the potential effects on root growth distribution in soil and its relationship with the accessibility of soil water.

We conducted a two-year field experiment on the Loess Plateau to examine the influence of plastic film mulching on the photosynthesis, respiration, leaf anatomy, root function,and yield of two maize cultivars planted at different densities.The main objectives of this study were to (1) investigate the physiological and anatomical responses of maize plants to mulching and (2) identify mechanisms associated with leaf and root function that increase crop yield in the mulching system.

2. Materials and methods

2.1.Experimental site description

Field experiments were conducted in 2017 and 2018 in the Yuanzhou district of Ningxia (36°18′N, 106°22′E; elevation 1769 m above sea level) in a hilly area of the Loess Plateau in northwest China. The annual average precipitation in this region is approximately 400 mm,approximately 60% of which occurs during July-September. The number of sunshine hours, daily mean air temperature, and frost-free period are respectively 2518.3 h,6.2°C and 151 days.The soil type at the experimental site is light loam, with 1.27 g cm?3soil bulk density,7.86 g kg?1organic carbon(C)content,0.81 g kg?1total nitrogen (N), 55.6 mg kg?1available N, 12.8 mg kg?1available phosphorus (P), and 158.0 mg kg?1available potassium (K) in the 0-40 cm soil layer.

2.2.Experimental design and crop management

A split-split plot design with three replications was established in the field. The main plots, subplots and subsubplots were plastic film mulching treatments, planting densities, and cultivars, respectively. Two plastic film mulching treatments were used:(1)ridge-furrow construction with alternating wide (80 cm) and narrow (40 cm) ridges and full plastic film mulching(hereafter referred to as FM;Fig.S1-A);and(2)as for FM but without mulching(hereafter referred to as RF; Fig. S1-B). The plastic film normally covers the soil surface to prevent water evaporation until maize maturity.Two maize cultivars, Zhengdan 958 (ZD958) and Xianyu 335(XY335),widely grown in China were used.Both cultivars were planted at two planting densities: low (7.5 × 104plants ha?1)and high (10.5 × 104plants ha?1). The low planting density is currently recommended in many regions of China, whereas the high planting density is associated with higher yield [35].Different cultivars and planting densities were used to gain a better understanding of the response of maize to plastic film mulching at the physiological level. There were eight treatments:(1)FMZL-ZD958,planted at low density under FM;(2)FMZH - ZD958 at high density under FM; (3) FMXL - XY335 at low density under FM;(4)FMXH-XY335 at high density under FM;(5)RFZL-ZD958 at low density under RF;(6)RFZH-ZD958 at high density under RF; (7) RFXL - XY335 at low density under RF;and(8)RFXH-XY335 at high density under RF.Plots of each treatment were 12.0 m×9.6 m in size and consisted of 16 rows 12 m long.A 1-m boundary of each plot was excluded during sampling and measuring.

A tractor-powered rotary harrow and leveling implements were used to prepare the land. Nine days before sowing,fertilizer application, ridging and plastic film mulching were performed. Two seeds were placed in a hole of 5 cm depth at the furrow bottom on April 26, 2017 and April 27, 2018 either under 12 μm thick and 120 cm wide transparent polyethylene for the FM treatment or with no mulching (in bare ground) for the RF treatment. About 1 month after sowing,one seedling was retained in each hole. Base fertilizer was applied at a rate of 123 kg N ha?1, 138 kg P2O5ha?1, and 75 kg K2O ha?1using urea,diammonium phosphate and potassium sulfate. An additional 200 kg N ha?1and 69 kg P2O5ha?1was applied using urea and diammonium phosphate during the growth season to prevent nutritional stress. The additional fertilizer was applied at the jointing stage with a maize dibbling device to perforate and fertilize between plants as deeply as possible. Weeds, pests and diseases were artificially controlled according to local management practices. In both years, the maize crop relied on natural precipitation for water. Maize plants were harvested on October 1, 2017 and October 3, 2018.

2.3. Sampling and measurement

2.3.1. Comprehensive meteorological drought index (CI)

CI values were collected from the National Climate Center of the China Meteorological Administration (https://cmdp.ncccma.net/influ/dust.php?dateStr=2017-4#calendar). These values were generated by calculating the precipitationnormalized index for the last 30 days (monthly scale) or 90 days(seasonal scale)and relative wetness index for the last 30 days. The CI value reflects not only climate anomalies but also crop water deficit and can thus be used to monitor drought conditions [36]. According to the national standard and local conditions in Ningxia, CI ≤?2.4 indicates excessive drought, ?2.4 ＜CI ≤?1.77 severe drought, ?1.77 ＜CI ≤?1.09 moderate drought, ?1.09 ＜ CI ≤ ?0.45 mild drought, and CI ＞?0.45 no drought. The CI value was used as an indicator of drought.

2.3.2. Soil water content and temperature

Soil water content (SWC) was measured in the 0-90 cm soil layer using a handheld steel soil auger at silking, grain filling and maturity stages under the FM and RF treatments. Soil samples were collected at three randomly selected points in each plot, packed in aluminum boxes, and brought to the laboratory After weighing they were dried at 105°C for at least 24 h.SWC was calculated as

The temperature of the topsoil under the FM and RF treatments was monitored with EasyLog USB data loggers(Lascar Electronics, London, UK) at half-hour intervals. Three loggers were buried at a depth of 20 cm in each plot during the growing season.

2.3.3.Maize yield determination

To determine grain yield, plants at physiological maturity were manually harvested from a 12-m2area in each plot,excluding border rows. Maize grain and biomass yield were determined with 14% moisture content and dry weight,respectively. Ear characteristics, including the length of barren ear tips, were measured on 15 randomly selected plants in each plot.

2.3.4. Gas exchange, chlorophyll fluorescence, and pigment content

At silking, grain filling, and maturity stages, three representative maize plants were selected from each plot, and net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO2concentration (Ci)were measured with a LI-6400 portable photosynthesis system (LI-COR, Lincoln, NE, USA) equipped with an LED leaf chamber, during 9:00-11:00 AM on a sunny,cloudless day.To determine dark respiration at night, gas exchange measurements were conducted from 11:00 PM to before sunrise the next day,and a larger leaf cuvette(2 cm×6 cm)was used with the LI-6400 gas analyzer to measure CO2efflux.To determine dark respiration during the day,leaves were dark-adapted for at least 30 min in advance. Ear leaves were sampled at the silking,grain filling,and maturity stages.

Chlorophyll fluorescence was measured during the same time period as Pnat all three growth stages. After maize ear leaves were dark-adapted for at least 30 min, the maximum photochemical efficiency (Fv/Fm) and non-photochemical fluorescence quenching (NPQ) were measured at the middle part of the ear leaf using a FluorPen portable fluorometer FP100(Proton System Instruments,Brno,Czech Republic).

Chlorophyll and carotenoid contents were measured spectrophotometrically according to a published protocol[37]. Ear leaves were ground in liquid N2and incubated with 100% acetone in darkness. After pigments were extracted from the leaf samples,pigments were quantified using a dualbeam spectrophotometer GENESY 10S UV-Vis(Thermo Scientific, Waltham, MA, USA). All procedures were conducted under low-light conditions, and samples were covered to minimize chlorophyll degradation caused by light. Three biological replications were performed for each treatment.

2.3.5.Leaf anatomy analysis

Stomatal density and aperture area were measured at the grain filling stage using colorless nail polish evenly applied to the abaxial surface of the ear leaf to form a replica of the leaf surface [38]. These imprints were observed under a light microscope DM5500B (Leica Microsystems, Biberach, Germany). Stomatal density was determined by counting the stomata in 18 fields of view per treatment.Ten stomata were randomly selected from images captured under the microscope (3 samples × 10 stomata = 30 stomata per treatment),and then stomatal aperture area was measured using ImageJ software(National Institutes of Health,Bethesda, MD,USA).

The middle part of the ear leaves (15 mm × 5 mm) was sampled from three representative maize plants at the grain filling stage. Samples were fixed in FAA solution (38% formaldehyde:glacial acetic acid:70% alcohol = 5:5:90, v/v) at 4°C and embedded in paraffin wax.Transverse sections were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems, LKB-II, Germany), stained with Fast Green,and counterstained with safranin. Leaf cross sections 8 μm thick were prepared under a light microscope DM5500B(Leica Microsystems, Biberach, Germany). Leaf anatomical traits were measured with ImageJ software (National Institutes of Health,Bethesda,MD,USA).

2.3.6. Root sap bleeding rate and root morphology measurements

At silking, grain filling, and maturity stages, the rate of root sap bleeding was determined as described previously [39],with slight modifications to adapt the method to maize stems.Three representative maize shoots were cut 10 cm above the ground in each plot.A small plastic bag containing cotton was used to cover each stem with the cotton in full contact with the incision,from 8:00 PM to 8:00 AM the next day.The plastic bag was closed tightly to prevent water loss and exclude dust and insects. Assuming that the exudate specific gravity was 1.0,the exudate volume was estimated based on the increase in cotton weight.The rate of root sap bleeding was estimated as the sap volume per hour per root (mL h?1root?1) [40].

Root morphology was measured at maturity.Roots of three representative plants were dug out of the 0-20 cm soil layer with their shape unchanged to measure the inter-plant and inter-row root angles.Inter-plant angle is defined as the angle between plants within a row and inter-row angle as the angle perpendicular to inter-plant root angle.Each root was cut at a depth of 10 cm and placed on a table such that it did not change the root morphology. Next, the length and width of the root projection on the table were measured to calculate the root angle using a trigonometric function. To measure root length and root dry weight, clods of soil were excavated at a depth of 60 cm with a steel shovel.The length and width of each clod were equal to the inter-row and inter-plant(within a row) spacing, respectively, with roots taken as the center, and each 20 cm layer was placed in a mesh bag. The roots were rinsed with water.To measure root morphology in different soil layers, roots were arranged in a shallow glass dish (25 cm × 20 cm) containing water and scanned with an Epson Perfection V800 Photo scanner (Seiko Epson, Naganoken,Japan).Root length in each soil layer was measured using WinRHIZO software (Regent Instruments, Quebec, Canada).After determination of the above indexes,roots were dried in an oven at 80°C to constant weight,which was recorded[41].

2.4. Statistical analysis

Analysis of variance and correlation were calculated using SPSS 12.5 (SPSS, Chicago, IL, USA), separately for each sampling event. Means were compared using Duncan’s multiple range test at P ＜0.05.

3. Results

3.1.Climate, soil water content, and topsoil temperature

Over the entire growing season (April-September), total radiation, mean air temperature, and total precipitation were respectively 3134.7 MJ m?2, 17.54 °C, and 387.50 mm in 2017 and 3069.1 MJ m?2,16.97°C,and 552.50 mm in 2018(Fig.1-A,B).Rainfall was greater in 2018 than 2017, especially between June 25 and July 10, 2018. The CI during June-August was much higher in 2018 than in 2017(Fig.2-A,B),indicating that 2017 was drier than 2018. Maize plants suffered from mild and serious drought (CI -1.8 to ?0.5) during July 17-24, 2017 at the silking stage (Fig. 2-A). Because of drought conditions, the CO2assimilation rate could not be measured, as leaf stomata were closed after 10:00 AM on a clear day.Accordingly,photosynthetic parameters were not measured during the 2017 growing season.

During the silking and filling stages in 2017, the FM treatment increased the water content of the 0-90 cm soil layer by 12.6%-28.0% and 12.0%-34.1%, respectively. However,at the maturity stage, there was no significant difference in water content at 30-90 cm between FM and RF(Fig.S2).During the silking and filling stages in 2018, the FM treatment increased the water content of the 0-90 cm soil layer by 1.4%-19.9% and 9.7%-25.8%, respectively, while at maturity, the water content in the 60-90 cm soil layer under FM treatment was lower than that under the RF by 3.3%-16.0%(Fig.S3).

From the sowing to jointing stage, jointing to silking,silking to grain filling, and grain filling to maturity, the mean daily temperatures of topsoil under FM treatment conditions were 2.4,1.1,1.3,and 1.3°C higher than RF treatment in 2017,respectively. In 2018, they were increased by 2.1, 0.3, 0.3, and 0.5°C,respectively(Fig.S4).

3.2.Grain and biomass yields

The FM treatment increased the grain yield of both maize cultivars. Under the FM treatment, the yield of ZD958 at low(ZL) and high (ZH) planting densities and XY335 at low (XL)and high (XH) planting densities was greater than that under the RF treatment by respectively 39.3%, 46.4%, 31.1%, and 34.7% in 2017, and by 21.5%, 3.6%, 13.1%, and 34.7% in 2018.The numbers of kernels per ear in ZL, ZH, XL, and XH were 8.2%-18.3% higher under FM than under RF in both years.The length of barren ear tips was significantly lower for the FM than RF(Fig.S2).The FM treatment significantly increased the 1000-kernel weight of cultivar ZD958 compared with XY335 in 2017. However, there was no difference in the 1000-kernel weight of XY335 between 2017 and 2018 under the FM treatment. In 2018, the FM treatment significantly increased the grain yields of ZL and XH(Table 1).Compared with RF,the FM treatment significantly increased biomass yields of ZL,ZH,XL,and XH by 15.4%-23.8% in 2017,and significantly increased those of ZH and XL by respectively 26.8% and 24.6% in 2018.The FM treatment significantly increased harvest index of ZL and ZH by 6.7% and 7.3%, respectively (Table S1). High planting density significantly increased the yields of both cultivars under both FM and RF treatments in 2017.However,high planting density significantly decreased the yield of XY335 under RF treatment in 2018 (Table 1). The analysis of variance showed that both FM and the year significantly affected grain yield, with the planting density showing no significant effect on grain yield. There was no interaction between FM and planting density,indicating that the effect of the plastic film mulching did not change with planting density. There was no interaction between FM and year in the regulation of grain yield(Table S3).

Fig.1-Meteorological radiation,air temperature,and precipitation at the experimental site in a semi-arid region of the Loess Plateau,China.Values collected in the maize growing seasons of 2017(A)and 2018(B)are plotted.

Fig.2-Comprehensive meteorological drought index(CI)at the experimental site in the semi-arid region of the Loess Plateau.Values of CI in the maize growing seasons of 2017(A)and 2018 (B)are plotted.

Table 1-Yield and yield components of maize in 2017 and 2018.

3.3. Photosynthesis and dark respiration

To identify the effects of plastic film mulching on crop yield,it is necessary to examine biochemical and photochemical processes such as photosynthesis and respiration that are critical for crop growth and development.At the silking stage,the Pnof ZL and XL was significantly higher under the FM than the RF treatment;at the grain filling stage,the Pnof ZH and XH was significantly higher under FM than under RF(Fig.3-A).Gsshowed a similar trend to Pnfor the FM and RF treatments(Fig.3-B). Compared with RF, the FM treatment showed significantly higher Trat both silking and grain filling stages.The Trwas higher for XY335 than ZD958 at both stages (Fig. 3-C),possibly because leaves with high photosynthetic capacity require high transpiration capacity.Although the Ciof ZL and ZH was 74% and 37% higher for the FM than for the RF treatment at the silking stage, respectively, there were no significant difference in Cibetween RF and FM at other growth stages(Fig.3-D).Compared with the low planting density,the high planting density significantly reduced Pn,Gs,Tr,and Ciof both cultivars at silking stage under FM(Fig.3).At silking,the Fv/Fmof both cultivars was slightly higher under FM than under RF, however, no significant differences were detected between the two treatments at the grain filling and maturity stages (Fig. 4-A). The NPQ of ZL, ZH, XL, and XH was lower at the silking and higher at the grain filling and maturity stages under FM than under RF. The NPQ of XY335 differed significantly between RF and FM at both silking and maturity stages (Fig. 4-B). Mulching affected NPQ in three growth stages,while planting density significantly affected NPQ only at the silking stage.Notably,the dark respiration rates of both cultivars during day and night were significantly higher for the FM than for the RF treatment at the silking and grain filling stages. Overall, the dark respiration rates of both cultivars were lower under high planting density than under low planting density under FM or RF conditions(Fig.5-A,B).

Analysis of variance showed that both FM and planting density affected gas exchange parameters. There was an effect of interaction between the FM and planting density on the regulation of stomatal conductance, transpiration rate,and dark respiration rate at maturity.FM and planting density also interacted in the regulation of the net photosynthetic rate during the silking and filling stages(Tables S4 and S5).

3.4.Pigment contents

In general, contents of chlorophyll a, chlorophyll b and total chlorophyll in maize ear leaf were higher under the FM than under the RF treatment at the silking and grain filling stages;however, there was no significant difference between RF and FM at maturity (Fig. 6-A-C). Plastic film mulching, cultivar,planting density and growth period did not affect the chlorophyll a/b ratio during the progression from silking to maturity(Fig.6-D).The carotenoid contents of neither cultivar showed significant differences between the RF and FM treatments (Fig. 6-E). The total chlorophyll and carotenoid contents were higher in XY335 than in ZD958 (Fig. 6-A-C). As plant density increased, total chlorophyll and carotenoid contents were not significantly changed at silking and filling stages. At the maturity stage, the cultivar XY335 showed a significant decrease in chlorophyll content under high planting density treatment.

Fig.3- Photosynthetic characteristics of maize ear leaves in 2018.(A)Photosynthetic rate.(B)Stomatal conductance.(C)Transpiration rate.(D)Intercellular CO2 concentration.Values represent mean±standard deviation.Different lowercase letters indicate significant differences among treatments at P ＜0.05 using Duncan's multiple range test.FM,ridge-furrow construction with full plastic film mulching; RF,ridge-furrow construction without mulching;ZL,ZD958 planted at low density(7.5×104 plants ha?1);ZH,ZD958 planted at high density(10.5×104 plants ha?1);XL,XY335 planted at low density(7.5×104 plants ha?1);XH,XY335 planted at high density(10.5 ×104 plants ha?1).

Fig.4-Maximum photochemical efficiency(Fv/Fm)and non-photochemical fluorescence quenching(NPQ)of maize ear leaves in 2018.(A)Fv/Fm.(B) NPQ.Values represent means±standard deviation.Different lowercase letters indicate significant differences among treatments at P ＜0.05 using Duncan's multiple range test.FM,ridge-furrow construction with full plastic film mulching;RF,ridge-furrow construction without mulching;ZL,ZD958 planted at low density(7.5× 104 plants ha?1); ZH,ZD958 planted at high density(10.5× 104 plants ha?1); XL,XY335 planted at low density(7.5× 104 plants ha?1);XH,XY335 planted at high density(10.5×104 plants ha?1).

Analysis of variance showed that FM significantly increased the total chlorophyll content at three growth stages,while the planting density had a significant effect on it only at the grain filling and maturity stages.The interaction between FM and planting density on total chlorophyll content was detected only at the filling stage(Table S6).

3.5. Leaf anatomy

Leaf anatomy changed under the FM compared to the RF treatment. Various leaf anatomical parameters, including thicknesses of the leaf and mesophyll layer; cross-sectional areas of the large vascular bundle, xylem, substomatal chamber and intercellular canal; and the ratio of intercellular canal cross-sectional area to large vascular bundle area were significantly lower for the FM than for the RF treatment;however, other parameters such as the lower epidermis thickness and phloem cross-sectional area did not significantly differ between FM and RF treatments(Table 2,Figs.S3,S4).The reduction in leaf thickness under the FM treatment was associated with a reduction in many traits including mesophyll thickness. However, the ear leaf stomatal densities of ZL, ZH,XL,and XH were significantly higher for the FM than for the RF treatment by 15.4%,12.7%,15.0%,and 17.4%,respectively(Table 2, Figs. S5, S6). Stomatal aperture areas of ZL, ZH, XL, and XH were higher for FM than for RF by 20.9%, 10.5%, 42.9%, and 14.3%, respectively. Under FM, the mean increase in stomatal aperture area was large (31.9%) at the low planting density, in comparison with 12.4% at the high planting density (Table 2,Figs. S5, S6). The increase in stomatal density under the FM treatment was independent of cultivar and planting density.

Analysis of variance showed that both FM and planting density significantly affected the thicknesses of leaf, mesophyll and upper epidermis, and the areas of stomatal aperture, substomatal chamber, large vascular bundle and xylem. There was no interaction between the two factors in the regulation of leaf thickness, mesophyll thickness and upper epidermal thickness(Table S7).

3.6.Root morphology and bleeding sap

Root morphology was affected by root angle (Fig. 7-A, B), which plays an important role in nutrient absorption. There was no significant difference in the inter-plant root angle between the FM and RF treatments;however,the inter-row root angles of ZL,ZH, XL, and XH were significantly greater for FM than RF by 24.0%,41.3%,30.9%,and 20.8%,respectively.The inter-plant root angle of XY335 was greater than that of ZD958(Fig.7-C).

Fig.5-Dark respiration rate of maize ear leaves in 2018.Dark respiration rates during the day(A)and night(B)are shown.Values represent means±standard deviation.Different lowercase letters indicate significant differences among treatments at P ＜0.05 using Duncan's multiple range test.FM,ridge-furrow construction with full plastic film mulching;RF,ridge-furrow construction without mulching;ZL,ZD958 planted at low density(7.5×104 plants ha?1);ZH,ZD958 planted at high density(10.5×104 plants ha?1);XL,XY335 planted at low density(7.5×104 plants ha?1);XH,XY335 planted at high density(10.5×104 plants ha?1).

Compared with RF, root dry weight was slightly higher in the 0-20 cm soil layer but significantly lower in the 20-40 cm and 40-60 cm layers under the FM treatment. The pattern of change in root dry weight in the 0-60 cm layer was similar to that in the 0-20 cm layer(Fig.8-A).Root length in the 0-60 cm soil layer was significantly greater under the RF than under the FM treatment at low planting density (Fig. 8-B). The proportion of root dry weight and length in the 0-20 cm soil layer was higher for the FM than for the RF treatment(Fig.8-C,D). The proportion of root dry weight and length in the 0-20 cm soil layer was higher for XH than ZH. The high planting density reduced the proportion of root dry weight and length of ZD958 but not XY335 in the 0-20 cm soil layer(Fig. 8-C, D). The rate of root sap bleeding was slightly higher for the RF than the FM treatment at both silking and grain filling stages. However, at maturity, the rate of root sap bleeding of ZL and XL was significantly higher for FM than RF by 269.6% and 512.1%,respectively (Fig.8-E).

Analysis of variance showed that FM significantly affected the inter-row root angle and planting density significantly affected the inter-plant root angle, but there was no effect of interaction between the two factors on root angle regulation.Both FM and planting density had a regulating effect on root dry weight,root length and the rate of root bleeding sap.There was an interaction effect between the two factors on the regulation of 20-60 cm root dry weight,root length,and rate of root sap bleeding at maturity(Table S8).

3.7.Correlation

Stomatal aperture area,net photosynthetic rate at the silking stage,and dark respiration during the day at three stages were significantly and positively correlated with maize grain yield.Only non-photochemical quenching at silking stage was significantly and negatively correlated with maize grain yield. Stomatal density, net photosynthetic rate at filling and maturity stages, dark respiration rate at night, maximum photochemical efficiency, non-photochemical quenching at filling and maturity stages, and chlorophyll content showed no correlation with maize grain yield(Table 3).

4. Discussion

4.1. Plastic film mulching increases maize yield potential in both dry and rainy seasons

Fig.6-Pigment contents of maize ear leaves in 2018.(A)Chlorophyll a content.(B)Chlorophyll b content.(C)Total chlorophyll content.(D)Chlorophyll a/b ratio.(E) Carotenoid content.Values represent means± standard deviation.Different lowercase letters indicate significant differences among treatments at P ＜0.05 using Duncan's multiple range test.FM,ridge-furrow construction with full plastic film mulching;RF,ridge-furrow construction without mulching;ZL,ZD958 planted at low density(7.5× 104 plants ha?1); ZH,ZD958 planted at high density(10.5×104 plants ha?1); XL,XY335 planted at low density(7.5× 104 plants ha?1);XH,XY335 planted at high density(10.5×104 plants ha?1).

Plastic film mulching with ridge-furrow planting increases maize growth and biomass, thus increasing crop yield because of increased topsoil temperature and moisture and decreased soil evaporation [16,22,24,32,42]. In arid and semiarid regions, such as the Loess Plateau of China, crop cultivation relies largely on natural precipitation. Crop yield is governed by the synchronization of plant growth with rainwater supply. However, increase in crop yield with the use of plastic film mulching is greater in areas with 200-450 mm of annual precipitation. For precipitation exceeding 450 mm, the effect of mulching on crop yield diminishes [13,43]. In the present study, the total precipitation levels at the experimental site were respectively 387.50 mm and 552.50 mm during the 2017 and 2018 growing seasons. The FM treatment significantly increased grain yields of ZL and XH under higher rainfall in 2018. This finding suggests that plastic film mulching increased maize yield under a wide range of precipitation conditions in the Loess Plateau. Mulching also significantly increased the biomass and grain yield of both cultivars at both planting densities in 2017. In a previous study [44], soil evaporation was significantly reduced and transpiration was increased under high planting density, increasing water use efficiency and yield [45]. This may be the reason for the increased yield under the higher planting density in the dry year 2017 (Table 1). However, maize grown at the high planting density displayed no significant increases in grain yield of either cultivar, compared to the low planting density under FM or RF treatment in 2018 (Table 1, Table S1), possibly owing to the decreased photosynthesis and transpiration (Fig. 3-A, C).Considering economic benefit, the high planting density(10.5 × 104plants ha?1) is not recommended in a relatively rainy year in this area.

Table 2-Anatomical indexes of maize ear leaves at the grain filling stage in 2018.

Fig.7-Inter-plant and inter-row root angles of maize plants at maturity.Image of maize roots showing the inter-plant angle(A)and inter-row angle(B) under different treatments.(C)Quantification of inter-plant and inter-row root angles under different treatments.Values represent means±standard deviation.Different lowercase letters indicate significant differences among treatments at P ＜0.05 using Duncan's multiple range test.FM,ridge-furrow construction with full plastic film mulching;RF,ridge-furrow construction without mulching;ZL,ZD958 planted at low density(7.5×104 plants ha?1);ZH,ZD958 planted at high density(10.5 ×104 plants ha?1);XL, XY335 planted at low density(7.5×104 plants ha?1); XH,XY335 planted at high density (10.5× 104 plants ha?1).

4.2. Increased stomatal density and stomatal aperture by plastic film mulching led to increased photosynthetic capacity and respiration

Crop productivity represents the balance between two important biological processes:photosynthesis and respiration[46].Studies have shown that plastic film mulching increases photosynthesis.Plastic film mulching increased soil moisture content and temperature [42], both of which have positive effects on photosynthesis [22]. Increased soil water content increases CO2diffusion from the atmosphere to the site of carboxylation because of greater stomatal aperture area and mesophyll conductance, and the greater stomatal aperture area and mesophyll conductance further increases Pn[47,48].In the present study, chlorophyll content, Pnand Gswere significantly higher under the FM than under the RF treatment at the silking and filling stages(Figs.3,6),in agreement with results of previous studies [21,22]. Leaf respiration is fundamental to sustaining several metabolic and physiological processes in plant canopies.However,the effect of plastic film mulching on leaf respiration remains unknown. In the present study,dark respiration during both day and night was increased under the FM treatment (Fig. 5). Higher respiration rates of tomato plants grown under elevated CO2 conditions were associated with increased leaf carbohydrate concentrations [49]. Respiration plays an important role in the maintenance of optimal rates of photosynthesis [50], because dark respiration provides ATP and converts carbohydrates to C skeletons needed for growth[51].An increase in soil moisture,ambient temperature, and photosynthetic supply of substrates led to increased leaf dark respiration [52]. Thus, the increased respiration rate of maize could have resulted from the coordinated process of both increased photosynthesis and increased soil water content under the FM treatment. Plastic film mulching increases the accumulation of carbohydrates[21,24], consequently increasing dark respiration. In the present study, Pn at the silking stage and dark respiration during the day at all stages were strongly positively correlated with maize grain yield (Table 3). In addition, the Pn at silking and grain filling stages and dark respiration rate in the day at maturity stage showed strong positive correlations with biomass yield(Table S2).Higher dark respiration rate provides not only more chemical energy for plant growth and development but also more C skeletons for the synthesis of important substances [53] beneficial for grain filling and decreasing the length of barren ear tips (Fig. S2). The above results further suggested that photosynthesis and respiration are highly coordinated to sustain plant growth and development under plastic film mulching. It should be noted that in the present study the leaf net photosynthetic rate was measured only during the morning and the dark respiration rate during morning and night,so net photosynthetic rate and respiration rate could not be scaled up to the whole day and growing season.

Fig.8- Root morphology and activity of maize plants under different treatments.(A-D)Root morphological parameters including root dry weight(A),root length(B),proportion of root dry weight(C)and proportion of root length(D)measured at maturity. (E)Rate of root bleeding sap measured at the silking,grain filling,and maturity stages.Values represent means±standard deviation.Different lowercase letters indicate significant differences among the treatments at P ＜0.05 using Duncan's multiple range test.FM,ridge-furrow construction with full plastic film mulching;RF,ridge-furrow construction without mulching;ZL,ZD958 planted at low density(7.5×104 plants ha?1);ZH,ZD958 planted at high density(10.5×104 plants ha?1);XL,XY335 planted at low density(7.5× 104 plants ha?1); XH,XY335 planted at high density(10.5 ×104 plants ha?1).

Table 3-Correlation of maize grain yield with gas exchange and chlorophyll fluorescence characteristics at three growth stages.

The Fv/Fmvalue is an indicator of leaf photosynthetic efficiency, while NPQ allows the dissipation of excess absorbed light energy as heat. The process of NPQ is directly or indirectly involved in several processes including light harvesting via the photosynthetic antenna complexes, transfer of captured energy to reaction centers, electron transport,proton translocation across the cell membrane, ATPase activity, and C assimilation [54]. In the present study, the Fv/Fmvalue increased at the silking stage for the FM compared with the RF treatment; however, NPQ showed the opposite trend (Fig. 4). Higher Fv/Fmand lower NPQ under the FM treatment indicated higher activity of photosystem II and transfer of more energy to reaction centers, resulting in increased photosynthesis. This result was supported by the strong correlation between NPQ and grain yield or biomass yield(Table 3, Table S2).

Leaf anatomy acclimates to the environmental conditions and plays a key role in CO2assimilation[55-57].The increased Pnand Gscould be explained by the significant increase in stomatal density and stomatal aperture area under FM conditions (Table 2, Figs. S5, S6), which may benefit CO2diffusion from the ambient air into plant leaves [57-59]. A strong positive correlation was detected between stomatal aperture area and grain yield (Table 3), suggesting that improved photosynthesis and respiration rate could be determined by stomatal behavior under FM conditions.Stomata control the supply of CO2for photosynthesis and evaporation of moisture for transpiration. The increased density and aperture area of stomata led to not only higher rates of net photosynthesis but also higher transpiration via the opened stomata[60].In the present study,maize plants in the RF treatment showed lower stomatal density and stomatal aperture area than in the FM, possibly because maize plants grown without mulching suffered from drought stress due to increased soil evaporation. A number of leaf anatomy parameters such as leaf thickness, mesophyll thickness and substomatal chamber area increased markedly in maize plants for the RF compared with the FM treatment. These modifications effectively prevented transpiration and represented an adaptation of plants to the arid climate [61]. The size of the substomatal chamber can adapt to promote CO2absorption[62].A larger substomatal chamber is beneficial for storing a greater amount of CO2, which may have alleviated the adverse effects of reduced stomatal density or stomatal aperture under the RF treatment.

4.3. Plastic film mulching modifies root distribution and activity

Previous studies [32,34,63] showed that plastic film mulching increases soil water availability and soil temperature, thus increasing various root growth parameters, including root length density, dry weight, surface area and volume, which further increase crop N and P uptake[22,33,64].In the present study,the inter-row root angle markedly increased for the FM compared to the RF treatment. Root dry weight was slightly increased in the 0-20 cm soil layer for the FM treatment;however,root length was significantly reduced in the 0-60 cm layer (Fig. 8). A possible explanation is that maize plants under the RF treatment suffered from drought stress at the early growth stage in 2018(before June 25),as shown by the CI value(Figs.1,2).Under water-deficit conditions in the field,a deeper and more extensive root system could help maize plants extract sufficient water and nutrients from the soil.

The rate of root sap bleeding is closely related to active water absorption by the root system and reflects the physiological root activity[65,66].Higher root physiological activity promotes biomass accumulation and increases yield [67]. In the present study, water retention and warming effect with the FM treatment significantly increased root activity at maturity(Fig.8), indicating that plastic film mulching could maintain and prolong root activity at the late growth stage.The increased root activity at maturity would ensure continuous availability of soil water and nutrients,which are critical for grain filling and yield formation.Taken together,our results indicate that plastic film mulching changed the root growth,which led to a modification of leaf anatomy. These changes in both root growth and leaf anatomy increased photosynthetic capacity and biomass production and thereby maize grain yield(Fig.9).

Fig.9-A schematic diagram showing the physiological and anatomical mechanisms of plastic film mulching to increase maize yield.

5. Conclusions

Crop management plays an important role in increasing maize production. Plastic film mulching has been used in maize production in the semi-arid areas of northwestern China for several decades.Plastic film mulching increased soil water content and temperature, and the water retention and warming effect increased inter-row root angle, proportion of root biomass in the 0-20 cm soil layer, and root activity at maturity. These changes in root morpho-physiology led to a modification of leaf anatomy, as indicated by increased stomatal density and aperture area of ear leaves under mulched conditions. The increased photosynthetic rate and dark respiration rate were due mainly to the higher stomatal density and aperture area of leaves.Net photosynthetic rate at silking and dark respiration rate in the day were significantly positively correlated with maize grain yield.

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 National Key Research and Development Program of China (2016YFD0300102). W.Z. was supported by the Innovation Program of the Chinese Academy of Agricultural Sciences and the Elite Youth Program of the Chinese Academy of Agricultural Sciences.

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.12.002.