Agricultural College,Shenyang Agricultural University,Shenyang 110866,P.R.China

Abstract In intercropping systems,high-positioned crops often exert shading stress on low-positioned crops,thus affecting the agronomic characteristics of the low-positioned crops.This study determined the mechanisms of photosynthetic,physiological and yield variations among peanut cultivars under shading stress.Four peanut cultivars,S60,C4,P12,and YS151,were grown in the field and subjected to shading stress for 77 days during reproductive stages.S60 and P12 showed lower yield and reduced biomass accumulation than C4 and YS151 under shading stress.Shading stress induced higher antioxidant enzyme activities in S60 and P12,relative to C4 and YS151.Under shading stress,S60 and P12 showed a higher lighttrapping capability than C4 and YS151,which was associated with changes in chlorophyll (Chl) a and b contents,and Chl a/b ratio.The net photosynthetic rate,stomatal conductance and transpiration rates of C4 and YS151 were decreased,but the intercellular CO2concentration increased under shading stress.The results demonstrated that non-stomatal limiting factors decreased the photosynthetic capacity of peanut under shading stress.The maximum photochemical efficiency of PSII(Fv/Fm) and non-photochemical quenching (NPQ) were higher in S60 and P12 than in C4 and YS151 under shading stress.These results suggest that S60 and P12 could absorb more light energy from weak light environments for photosynthesis than C4 and YS151 and dissipate the excess energy in the form of heat to improve their light protection ability.This study explains the inter-variety differences in shading stress tolerance in peanut and provides physiological parameters for guiding the selection of shade-tolerant cultivars.

Keywords:peanut,shading,antioxidant enzyme system,photosynthesis,fluorescence,yield

1.Introduction

Intercropping of leguminosae and gramineae crops is a crucial and common farming practice in the world.This cropping system can effectively exert the marginal effect of crops and nitrogen fixation by legumes,provide the maximum use of light and heat resources,and increase the production value of cultivated land (Xuet al.2008;Liet al.2019).However,due to differences in plant shape and height in intercropping systems,high-positioned crops exert shading stress on low-positioned crops,which affects the field microclimate of the low-positioned crops (Carrutherset al.2000;Lianget al.2019).Light is an important environmental factor in crop growth and development.Thus,shading stress interferes with plant growth and development,resulting in morphological changes (Wang X Cet al.2020),decreased photosynthetic capacity (Yaoet al.2017a) and changes in enzyme activity (Yaoet al.2017b).As the height of high-positioned crops increases,the severity of shading stress also intensifies,particularly during reproductive growth stages (Yaoet al.2017b;Wang X Cet al.2020).The reproductive growth stage is the key period for crop yield formation,and changes in environmental light conditions could severely impact crop yield and quality (Yanget al.2017;Liuet al.2018;Wang X Cet al.2020).Therefore,understanding the physiological response mechanisms of crops under shading stress is essential for the rational allocation of intercropping cultivars and improvement of crop yield.

Leaves are the plant organs that detect changes in the light environment first.Studies have shown that shading stress could reduce the photosynthetic rate of leaves,thus reducing photosynthetic product levels and decreasing biomass accumulation (Yaoet al.2017a;Liet al.2019).Under shade conditions,changes in the light environment could seriously impede the primary processes of photosynthesis,which include light energy absorption,excitation energy transfer,photochemical reaction,electron transfer processes,proton gradient establishment,ATP synthesis,and CO2fixation among others (Zhanget al.2004;Terashimaet al.2006;Jianget al.2011).Previous studies have shown that shading can block energy transfer from photosystem II(PSII) to PSI,reduce the thickness of leaves,palisade and spongy tissues and decrease chlorophyll fluorescence(Wuet al.2007;Yaoet al.2017b).Different cultivars have different photosynthetic responses to shade treatment.Thus,photosynthetic parameters can be used as screening indexes for crop shade tolerance traits.Yaoet al.(2017a)showed that under shade treatment,the shade-tolerant soybean cultivar L32 has higher photosynthetic capacity and yield due to PSII activity and energy transport from PSII to PSI,compared to the shade-sensitive soybean cultivar L29.Suet al.(2014) reported that photosynthetic parameters,such as leaf area index,chlorophyll (Chl) content and net photosynthetic rate (Pn),can be used to evaluate the shade tolerance levels of different soybean varieties.Changes in the activity of the PSI and PSII reaction centers in leaf chloroplasts caused by shading stress can lead to excessive production of reactive oxygen species (ROS)(Foyer and Noctor 2005;Ruban 2012;Liet al.2013)and impede cell growth and metabolism.Superoxide dismutase (SOD) (EC 1.15.1.1),peroxidases (POD) (EC 1.11.1.7) and catalase (CAT) (EC 1.11.3.6) are important antioxidant enzymes in plants (Xuet al.2011),which remove excessive ROS and maintain the activities of PSI and PSII reaction centers.Therefore,high photosynthetic capacity and antioxidant enzyme activities are important in resisting shade stress.

Peanuts (Arachis hypogaeaLinn.) are an important oil crop and suitable for planting in sandy soils with good drainage.In Northeast China,peanut growing regions typically experience strong monsoon winds,which often cause severe wind erosion.These effects not only cause significant loss of soil nutrients,but also occasional sandstorms and environmental pollution.The intercropping of sorghum (Sorghum bicolor(L.) Moench) and peanut is a common farming practice in this area,which plays an active role in preventing and controlling wind erosion (Chenet al.2010;Gronleet al.2015;Lianget al.2016).However,this intercropping system subjects peanut plants in the reproductive growth stage to shade stress from the sorghum,and negatively affects peanut yield.Previous studies on intercropping of peanut with sorghum focused on cultivar selection,optimization of farming patterns and agricultural production efficiency evaluation (Matthewset al.1991;Zuo and Zhang 2003;Gopalet al.2010;Xuet al.2016).However,differences in the physiological mechanisms and responses of different peanut cultivars to shading stress are still unclear.

In this study,the various photo-physiological parameters of four local peanut cultivars with similar maturity ages but different sensitivities to shading stress were analyzed to elucidate their photosynthetic responses under shade treatment.These parameters includedPn,stomatal conductance (gs),intercellular CO2concentration (Ci),transpiration rate (Tr),Chl content,PSII primary light conversion efficiency (Fv/Fm),PSII actual photochemical quantum yield (Yield (II)),electron transport rate (ETR),non-photochemical quenching (NPQ),and photochemical quenching coefficient (qP).We also examined the antioxidant capacities,biomass accumulation and yields of these four peanut cultivars under shading stress,and provide criteria for the selection of shading tolerance in peanut cultivars.

2.Materials and methods

2.1.Experimental design and treatment

The experimental site was located in Guyushu Town,Changtu County (42°78´N,124°10´E),Liaoning Province,an important peanut-production center in northern China.The area is in the north temperate continental seasonal sub-humid climatic zone.The rainfall and average daily temperatures from sowing to maturity were 380.5 mm and 23.5°C in 2015,and 410.0 mm and 22.5°C in 2016.The experimental site has sandy soil with the following properties:organic matter 8.9 g kg−1,available nitrogen 64.6 mg kg−1,available phosphate 28.1 mg kg−1,and available potassium 78.3 mg kg−1.This study used four peanut cultivars with similar growth periods,including S60,C4,P12,and YS151.Average-sized peanut seeds were sown on May 8,2015 and May 10,2016,at a planting density of 150 000 holes ha−1(60 cm×11 cm),with two seeds per hole and at a depth of 5 cm.After germination,seedlings were thinned to ensure only one plant in each hole.Fertilizer application and weed management were done according to standard field management methods.The experiment involved a shade treatment imposed during the flowering period (more than 50% of peanuts flowering;June 25,2015;June 27,2016),and a non-shade treatment was used as the control (CK).Shade was provided by setting up a shade shed in the field,which consisted of a square iron frame with a height of 1.5 m.The top of the shed was covered with a black shading net (50% shade compared to the natural light).Diurnal variations in photosynthetic photon flux density(PPFD) were measured throughout a day using a light meter(AccuPAR LP-80,USA) (Fig.1).The shade and non-shade treatments were separated by eight rows.Sampling and yield measurement plots were set up separately:24 plots for sampling,24 plots for yield measurement,for a total of 48 plots.A randomized block design with three replicates was established,and plants were planted in bare soil in a north-south row direction.The rows in the experimental plot were 5 m long,0.6 m wide,with five rows in each plot,and a plot area of 15 m2.The first day after shading stress (DAS)was recorded as 1 DAS.Plant height and root,stem,and leaf biomass were measured at 17,32,47,and 62 DAS in both 2015 and 2016.Leaf sampling and measurements of photosynthetic and fluorescence parameters were done at 17,37,57,and 77 DAS in 2016.

Fig.1 The photosynthetic photon flux density (PPFD) changes of different light levels within a day in 2015 (A) and 2016 (B).

2.2.Sampling

Mature leaves close to the newly expanded leaves were sampled between 9:00 and 11:00 a.m.for the evaluation of various physiological and biochemical parameters.Leaf samples were rapidly frozen in liquid nitrogen then stored at–80°C until use for the determination of Chl content,antioxidant enzyme activities and malondialdehyde (MDA)content.For biomass evaluation,the aboveground plant parts were cut at the cotyledon node and divided into two parts:stems and leaves.The roots were dug out with a spade and thoroughly rinsed to remove the soil.Samples were oven-dried at 105°C for 30 min and further dried to a constant weight at 80°C to measure the root,stem and leaf biomass.At maturity,all pods in each plot were harvested and air-dried until the water content fell below 9%.The weight of the pods was used to calculate the yield.We also determined the number of pods with a single kernel (i.e.,the number of partially filled pods with only one kernel) and double kernels,and evaluated the kernel ratio and the 100-kernel weight.

2.3.Measurement of gas exchange parameters

The third fully expanded leaf on each plant (from the bottom of the main stem) was sampled at 9:00–11:00 a.m.and used to quantify various photosynthetic parameters using an LI-6400 portable optical instrument (LI-COR Biosciences Inc.,Lincoln,NE,USA).The light intensity was set to 1 800 μmol (photon) m–2s–1;the air temperature was kept at 25°C,CO2concentration at 400 μmol mol–1and airflow at 500 μmol s–1.The evaluated gaseous exchange parameters includedPn,gs,Ci,andTr.

2.4.Measurement of fluorescence parameters

Chl fluorescence was measured by a PAM-2500 pulsemodulated fluorometer (Walz,Germany) and analyzed by the PAMwin3Software.Peanut leaves were dark-adapted for 30 min before measurement.A very low modulated light(<0.1 μmol (photon) m–2s–1) was used to determine the minimal fluorescence yield of the dark-adapted state (Fo).The maximal fluorescence of the light-adapted state (Fm)andFv/Fmwere determined by a 3 000-ms saturated light pulse.To determine the steady state (Fs) and the maximal fluorescence of the light-adapted state (Fm´),leaves were illuminated with an actinic light (1 800 μmol (photon) m–2s–1).Fv/Fmwas calculated following the method of Van and Snel(1990).The effective quantum yield of PSII photochemistry(ΦPSII) and ETR were calculated as described by Gentyet al.(1989).qPwas calculated using the equationqp=(Fm–Fs)/(Fm–Fo),while the NPQ was calculated by the equation:NPQ=(Fm–Fm´)/Fm´

2.5.Chl content

Total Chl was extracted according to Yaoet al.(2017b).Briefly,0.2 g of ground leaves were incubated in 10 mL anhydrous ethanol in the dark at room temperature for 24 h.The absorbance values of the supernatant at wavelengths of 470 and 648 nm were determined.Chl contents (Chla,Chlb,Chla+b,and Chla/b) were expressed as mg g–1FW and calculated according to Porra (2002).

2.6.Antioxidant enzyme activities and MDA concentration

SOD,POD and CAT were extracted according to Djanaguiramanet al.(2009).Frozen leaf tissue (0.2 g)was ground in 5 mL of 0.1 mol L–1Tris-HCl buffer (pH 7.8)containing 1% polyvinyl pyrrolidone,1 mmol L–1EDTA and 1 mmol L–1dithiothreitol.The homogenate was centrifuged at 18 000×g for 20 min at 4°C.Then,the supernatant was used for the measurement of enzyme activities.SOD,POD and CAT activities were assayed according to Leiet al.(2006),Chance and Maehly (1955) and Djanaguiramanet al.(2009),respectively.

MDA content was determined by the 2-thiobarbituric acid (TBA) test,as described by Havir and McHale (1987).

2.7.Statistical analysis

The data were analyzed by three-way analysis of variance(ANOVA) and Duncan’s multiple range tests using the SPSS Software (version 20,Chicago,IL,United States).The data presented are means (±SD) of three independent replications.All graphs were plotted using Sigmaplot(version 12,Systat Software).The correlations between peanut yield and physiological parameters were assessedviaPearson’s product-moment correlation (Duet al.2019).

3.Results

3.1.Effects of shading on peanut yield and yield traits

Three-way ANOVA revealed that genotype (G) and treatment (T) significantly affected the yield of four peanut cultivars.There were no statistically significant differences in the year (Y),G×Y,T×Y,T×G,or T×G×Y interactions for yield or yield traits,except Y for kernel ratio and T×G interaction for yield,double kernel pods number,single kernel pods number,and kernel ratio (Table 1).The yield and yield traits of the four peanut cultivars differed significantly under both control and shading conditions.Shading stress during reproductive stages decreased yield,the number of pods with double kernels,100-kernel weight,and kernel ratio.The yield reductions of C4 and YS151 were significantly higher than those of S60 and P12 under shade stress.For example,compared to the control,yield,double kernel pods number,100-kernel weight,and kernel ratio were reduced under shade stress by 10.57,13.74,2.71,and 6.38% in S60,22.52,33.91,6.29,and 17.87% in C40,15.72,13.40,2.83,and 8.11% in P12 and 31.20,38.51,4.90,and 16.43% in YS151.Compared to the control,shading stress significantly increased the number of pods with single kernels in all the four peanut cultivars,and the increments were higher in C4 (increased by 58.33%) and YS151 (79.17%) than that in S60 (21.74%) and P12 (18.52%) (Table 1).The yields and yield traits under shading stress in 2016 were similar to those in 2015.

3.2.Effects of shading on peanut plant height and biomass accumulation

Under non-shade conditions,peanut plant height increased with the growth period (Fig.2-A and B).The four peanut cultivars showed different plant height responses to shading stress at different growth stages.There were no significant differences in the heights of the four peanut cultivars,between control and shading stress at 17 DAS.For cultivars C4 and YS151,shading stress significantly increased the plant heights,relative to the control during 32–62 DAS.However,shading stress only significantly increased plant height observed at 47–62 DAS in S60 cultivar and 62 DAS in P12 cultivar.

The four peanut cultivars showed different biomass accumulation responses to shading stress at different growthstages (Fig.2-C–H).There were no significant differences in root biomass in S60 and P12 cultivars between the control and shading stress plants during the whole span of reproductive stages (Table 2).However,shading stress significantly decreased the root biomass of cultivars C4 and YS151,as compared to the control during 17–62 DAS,except that no significant difference was observed in YS151 at 17 DAS between the control and shading stress plants(Fig.2-C and D).Shading stress significantly decreased stem biomass accumulation compared to the control,as was observed at 62 DAS in S60 cultivar,32–62 DAS in C4 cultivar,47–62 DAS in P12 cultivar,and 17–62 DAS inYS151 cultivar (Fig.2-E and F).Compared with the control,leaf biomass accumulation was significantly decreased under shading stress at 62 DAS for S60 and P12 cultivars,and at 17–62 DAS for C4 and YS151 cultivars (Fig.2-G and H).The results of plant height and biomass accumulation under shading stress in 2016 were similar to those obtained in 2015 (Table 2).

Table 1 Effect of shading on peanut grain yield and yield traits in 2015 and 2016

3.3.Effects of shading stress on antioxidant activities and MDA contents in leaves

Relative to the control,the four peanut cultivars showed different antioxidant activities and MDA contents under shading stress at different growth stages (Fig.3).Shading stress significantly increased SOD,POD and CAT enzyme activities in peanut leaves during 17–77 DAS (Fig.3-A–C).Under shading stress,the SOD,POD and CAT enzyme activities in S60 and P12 were higher than those in C4 and YS151.The MDA content was increased by 22.39–30.23% in S60,50.82–60.82% in C4,22.22–30.23% in P12 and 45.71–59.78% in YS151 by shading stress,as compared to the control during 17–77 DAS (Fig.3-D).Genotype (G),treatment (T),growth stage (S),and their interactions significantly affected the activities of SOD,CAT and POD and the content of MDA in peanut leaves according to the three-way ANOVA (Table 3).

3.4.Effects of shading stress on Chl contents in leaves

The results of three-way ANOVA showed that G,T,growth stage (S),and T×G interaction significantly affected Chl content (Chla,Chlband Chla+b) and Chla/bratio in peanut leaves.The interactions between G×S,T×S and T×G×S did not significantly affect the Chl content or Chla/bratio,except G×S interaction for Chla/b(Table 3).Shading stress increased Chl content but reduced the Chla/bratio (Fig.4).Under shading stress,the increments of Chla,Chlband Chla+bcontents in S60 and P12 were more substantial than those in C4 and YS151.Meanwhile,the reductions in Chla/bratio in S60 and P12 were less apparent than those in C4 and YS151 (Fig.4;Table 3).

Fig.2 Effects of shading stress on peanut plant height and biomass accumulation in 2015 and 2016.A and B,plant height.C and D,root biomass.E and F,stem biomass.G and H,leaf biomass.Bars are mean±SD (n=3).*,significant at P<0.05.

Table 2 Analysis of variance of plant height and biomass accumulation in 2015 and 2016

Fig.3 Changes of superoxide dismutase (SOD) (A),catalase (CAT) (B) and peroxidases (POD) activities (C) and malondialdehyde(MDA) contents (D) in leaves of four peanut cultivars under control and shading stress conditions.Bars are mean±SD (n=3).*,significant at P<0.05.

3.5.Effects of shading stress on gas-exchange parameters in leaves

The gaseous-exchange parameters of the four peanut cultivars showed differential responses to shading stress under different growth stages (Fig.5).There were no significant differences ofPnin S60 or P12 cultivars between the control and shading stress plants during reproductive stages.However,Pnwas significantly decreased by 15.96–26.85% in cultivar C4 and by 15.28–20.46% in YS151,under shading stress as compared to the control during 17–77 DAS (Fig.5-A).

Thegsin the four peanut cultivars was significantly decreased under shading stress,as compared to the control during 17–77 DAS.However,no significant differences were observed in cultivar S60 at 17–37 DAS or in cultivar P12 at 17 DAS.Greater reductions ofgswere observed in C4(decreased by 14.71–31.48%) and YS151 (16.95–35.42%),relative to S60 cultivar (6.25–16.28%) and P12 cultivar(9.62–12.20%) under shading stress as compared to the control (Fig.5-B).

TheCiwas significantly decreased in the four peanut cultivars under shading stress,as compared to the control during the reproductive stages.However,no significant differences were observed in S60 at 17–57 DAS or in P12 at 17–37 DAS.Greater increments ofCiwere observed in cultivars C4 (increased by 4.37–7.97%) and YS151(4.54–8.43%),relative to cultivars S60 (1.10–2.65%) and P12 (1.67–2.89%) under shading stress,as compared to control (Fig.5-C).

No significant differences inTrwere observed between the control and shade-stressed plants in the four peanutcultivars at 17 DAS.In cultivars C4 and YS151,shading stress significantly decreasedTrcompared to the control during 37–77 DAS.However,shading stress only significantly decreasedTrobserved at 77 DAS in S60 and 57–77 DAS in P12 (Fig.5-D).Furthermore,G,T,S,and their interactions (T×G×S) significantly affectedPn,gs,Ci,andTrin peanut leaves according to the three-way ANOVA,with the exception that T×G×S interaction had no significant effect ongs(Table 3).

Table 3 Analysis of variance of physiological parameters

3.6.Effects of shading on Chl fluorescence parameters in leaves

Three-way ANOVA revealed that G,T and S significantly affectedFv/Fm,Yield (II),ETR,NPQ,andqPin leaves,except G for NPQ,T for NPQ andqP.There were no significant differences of T×G,G×S,T×S,or T×G×S interactions for Chl fluorescence parameters (Table 3).Fv/Fmwas significantly increased under shading stress by 13.10–21.13% in S60 and 14.67–17.50% in P12 compared to the control during 17–77 DAS.Meanwhile,no significant differences were observed in C4 or YS151.Shading stress had no significant effects on Yield (II),ETR orqPin the four peanut cultivars.NPQ was significantly increased under shading stress by 14.29–34.78% in S60 and 13.91–41.86% in P12 compared to the control during 17–77 DAS.However,no significant differences were observed in C4 or YS151 (Table 4).

3.7.Correlations between peanut yield and physiological parameters

Peanut yield showed significant positive correlations(P<0.05) withPnandTrduring 17–77 DAS andgsat 17,37 and 77 DAS.Peanut yield was negatively correlated withCiat 17,37 and 77 DAS,Chla+bat 17 DAS,SOD activity and MDA content during 17–77 DAS,and POD and CAT activities during 17–57 DAS (Table 5).There was no significant correlation between peanut yield and Chl fluorescence parameters.

4.Discussion

Changes in the light environment can affect plant morphogenesis,photosynthetic physiology,storage,and accumulation of nutrients and thus reduce the quality and quantity of crop yield (Yuet al.2015;Fanet al.2018;Chenet al.2019).Previous studies have shown that shading stress can reduce the yield of various crops,including soybean (Wang X Cet al.2020),spring maize (Wang Jet al.2020) and wheat (Wang Yet al.2020).Furthermore,the extent of crop yield reduction is related to the cultivar,shading intensity,shading duration,and shading degree.In this study,shading stress during the reproductive stage significantly decreased the yield of peanut cultivars(Table 1).Four peanut cultivars,S60 and P12 (shadingtolerant) and C4 and YS151 (shading-sensitive),were screened to evaluate the mechanisms of the shade stress response and cultivar-dependent shading stress tolerance in peanut.

Fig.4 Changes of chlorophyll (Chl) a (A),Chl b (B) and Chl a+b (C) contents and Chl a/b (D) in the leaves of four peanut cultivars under control and shading stress conditions.Bars are mean±SD (n=3).*,significant at P<0.05.

4.1.Regulation in biomass distribution contributes to peanut plant shading tolerance

The planting density,number of pods and grain weight are the basic elements of peanut yield.In this study,shading stress significantly decreased the number of pods with double kernels,100-kernel weight and kernel ratio of each of the four peanut cultivars,thus reducing peanut yield(Table 1).Furthermore,peanut cultivars S60 and P12 had lesser decreases in these yield components than C4 and YS151.The results indicate that S60 and P12 are more suitable for peanut and sorghum intercropping than C4 and YS151.In general,crops in intercropping systems compete for environmental resources such as living space,light,heat,water,and fertilizer.Previous studies have shown that the application of rational cultivation methods,crop allocation,cultivar selection,and planting density can improve the economic benefit of intercropping systems (Ghosh 2004;Agegnehuet al.2008;Dahmardehet al.2009).Therefore,understanding the mechanisms of shading tolerance in peanut and selecting shading-tolerant cultivars could benefit peanut and sorghum intercropping.

Fig.5 Changes of net photosynthetic rate (Pn) (A),stomatal conductance (gs) (B),intercellular CO2concentration (Ci) (C),and transpiration (Tr) (D) in leaves of four peanut cultivars under the control and shading stress conditions.Bars are mean±SD (n=3).*,significant at P<0.05.

The change in light intensity and light quality directly affects plant morphological characteristics and physiological and metabolic parameters (Liuet al.2017;Yanget al.2018).A previous study indicated that shading stress reduces photosynthetic parameters,such as effective photosynthetic radiation and red to far-red ratio (R/FR)in the crop canopy,resulting in a shade response of the crop (Wanet al.2015;Liuet al.2017;Yaoet al.2017a).Plants under shading stress show significant morphological changes,such as stem and petiole lengthening,branch reduction,early flowering,and leaf reduction (Wuet al.2017).Previous studies have shown that the shading stress caused by corn in corn-soybean intercropping systems increases soybean plant height and internode length of the stem,and thins the leaf stalk (Yanget al.2014;Yucelet al.2020).These morphological changes allow plants to absorb more light energy and evade shading stress.In the present study,shading stress significantly increased the heights of the various peanut cultivars (Fig.2-A and B).An increase in shading duration increased peanut plant height,severely affected plant morphology and increased the lodging vulnerability.Interestingly,although C4 and YS151 showed a greater rate of increase in height than S60 and P12 under shading stress,C4 and YS151 showed lower biomass accumulation and yield than S60 and P12 (Table 1;Fig.2).This result could be related to the management of photosynthate allocation under shading stress.Shadingsensitive peanut cultivars allocated more photosynthetic products to the elongation of the main stem to obtain more light (Fig.6).Reasonable distribution of biomass plays an essential role in improving crop yield.Previous studies have shown that shading can severely affect the biomass accumulation of soybean (Wang X Cet al.2020),maize(Gaoet al.2020) and wheat (Wanget al.J 2020),and ultimately affect crop yield.Shade treatment affects the shape and size of leaves (Gonget al.2014) due to weaker canopy light.For example,leaves become longer,wider and thicker with lower biomass.Changes in the light environment and leaf morphology can reduce the photosynthetic efficiency of plants,which leads to an insufficient supply of the energy needed for tissue and organ growth and metabolism (Fig.6).Taken together,this regulation of biomass distribution could be a regulatory mechanism for plants to adapt to lowlight environments.

4.2.Regulation of antioxidant defense system contributes to peanut plant shading tolerance

Previous studies have shown that shading stress can induce excessive ROS production in plants,causing oxidative damage,membrane peroxidation and metabolic disorders,which severely inhibit plant growth (Foyer and Noctor 2005;Ruban 2012;Liet al.2013).Plants have evolved highly sophisticated antioxidant defense systems,such as antioxidant enzymes (i.e.,SOD,POD and CAT) and non-enzymatic antioxidants (i.e.,GSH and AsA),to scavenge the redundant ROS induced by various environmental stresses (Garnczarskaet al.2004).SOD,POD and CAT are important antioxidant enzymes,as well as vital screening indicators for stress resistance.Numerous studies have shown that the activities of these antioxidant enzymes vary among cultivars,shading treatment durations,growth periods,and treatment intensities (Kwonet al.2006;Menget al.2007;Gill and Tuteja 2010;Penget al.2010;Du and Wang 2012).In the present study,shading stress increased the activities of SOD,POD and CAT in the leaves of all peanut cultivars,relative to the control (Figs.3 and 6).These results indicate that shading stress could induce oxidative damage to peanut plants.MDA,the primary product of membrane lipid peroxidation,is often used to evaluate the degree of damage to plant membrane systems and the stress resistance level of plants.In this study,shading treatment resulted in the accumulation of MDA in peanut leaves,and the MDA contents increased continuously with the extension of shading time (Fig.3-D).It is worth noting that S60 and P12 showed higher antioxidant enzyme activities than C4 and YS151,but lower MDA contents than S60 and P12 under shading stress (Fig.3).This result suggests that S60 and P12 had higher ROS scavenging capacities under shading stress than C4 and YS151,which could increase the tolerance to shading stress in S60 and P12 seedlings.

Table 5 Pearson’s correlations of peanut yield with physiological parameters

4.3.High efficiency of light energy absorption and photosynthetic capacities was beneficial to peanut plant growth under shading stress

Chl can absorb,transmit and transform light energy,which is an important component of the plant photosynthetic system(Kalajiet al.2012;Lazár 2015).Chlaandbare the main components of chlorophyll in higher plants.Chlais directly related to photosynthesis,while Chlbis mainly located in the photochromatin complex of photosystem II,which is responsible for trapping diffuse light (Fieldet al.2013).Shading stress increases the content of photochromic proteins in the photosynthetic system,which can bind more Chl and reduce Chl degradation and photooxidation,increasing the Chl content.An increase of Chl content in plant leaves,including Chla,Chlband Chla+b,is conducive to the absorption and capture of more light energy from low-light environments and for improving the efficiency of light energy utilization (Gill and Tuteja 2010;Yaoet al.2017b).In this study,shading stress significantly increased the contents of Chla,banda+bin leaves,and decreased the Chla/bratio (Figs.4 and 6).The decrease in Chla/bratio is mainly because shading stress induced a greater increase in Chlbcontent than that of Chla.Furthermore,S60 and P12 had higher Chl contents (Chla,Chlband Chla+b) than C4 and YS151 under shading stress,but lower Chla/bthan C4 and YS151 (Fig.4).The decrease in Chla/bvalue was beneficial for improving the low light utilization rate of peanut plants,which enhances the survival ability of peanut plants under shading stress.Taken together,shade-tolerant peanut cultivars can effectively adjust the proportion and distribution of Chl,increase the interception of diffuse light under weak light conditions and make better use of available light energy.

The leaf is the main photosynthetic organ,and shading stress can inhibit leaf growth and photosynthesis (Gill and Tuteja 2010;Yaoet al.2017b).In general,both stomatal restriction (such as light intensity,temperature and water) and non-stomatal restriction (such as O2,CO2and photosynthetic enzymes) can reduce the photosynthetic capacity of plants (Gonget al.2015).Zivcaket al.(2014)showed that shading stress decreased the photosynthetic capacity of spring barley by stomatal restriction.In the present study,shading stress decreased thePn,gsandTrof C4 and YS151 leaves,but increasedCi(Fig.5),suggesting that shading stress decreased the net photosynthetic rate of peanut leaves by non-stomatal limitation.This effect could be related to the regulation of Rubisco activities under shading stress (Xiaet al.1999;Zhanget al.2019).Interestingly,shading decreased thegsandTrof S60 and P12 and increasedCi,relative to the control.However,thePnof the control and shade-treated plants did not differ significantly.The results indicate that S60 and P12 had higher photosynthetic capacities under shading stress than C4 and YS151,which might contribute to shade stress tolerance in S60 and P12 seedlings.

Fig.6 The comprehensive model of physiological metabolism regulation in peanut plants under shading stress.Changing the light environment regulates the chlorophyll contents,and photosynthetic and antioxidant enzymes activities to adapt to the environmental stress.The distribution and regulation of photosynthetic products affect the agronomic characters and yield of peanut plants.Up-regulated items under shading stress are marked with upward red arrows,and down-regulated items under the shading stress are marked with downward green arrows.LHC,light harvesting pigment protein complexes;NPQ,non-photochemical quenching;qP,photochemical quenching coefficient;ROS,reactive oxygen species;SOD,superoxide dismutase;POD,peroxidases;CAT,catalase;APX,ascorbate peroxidase.

In general,the light energy absorbed by crops is consumed in three ways:(1) photosynthesis;(2) as heat energy;and (3) fluorescence quenching (Fig.6).Chl fluorescence is widely used in crop science as an intrinsic probe to study the relationship between environmental stress and photosynthesis (Schreiber and Bilger 1993).Changes in Chl fluorescence parameters reflect the adaptability of crops to different light environments.The Chl fluorescence of plants is mainly emitted by the Chlaof PSII,which reflects the activities of light energy absorption,transfer,dissipation,and distribution.Previous studies have shown that shading stress significantly affects the minimal fluorescence (Fo) of crops by influencing the content of Chl,which finally affects the maximal quantum yield of PSII (Fv/Fm) (Demmiget al.1987;Mauroet al.2011;Zhanget al.2018;Lianget al.2019).In the present study,shading stress increased theFv/Fmand non-photochemical quenching (NPQ) of S60 and P12 but did not have any significant effect on the quantum photosynthetic yield(Yield),apparent photosynthetic electron transfer rate(ETR),or photochemical quenching coefficient (qP) when compared with the corresponding control (Table 4;Fig.6).Furthermore,there were no significant differences in these Chl fluorescence parameters between the control and the shading treated C4 or YS151.These results suggest that the shade-tolerant peanut cultivars S60 and P12 probably absorb more light energy under shade stress than the shade-sensitive peanut cultivars C4 and YSL50,which ensures optimal photosynthetic rates.However,some of the light energy absorbed was not utilized by the plant but was dissipated in the form of heat energy.Such dissipation of radiation energy plays a key role in protecting the peanut plants from light damage.

5.Conclusion

This study revealed the differential mechanisms of shade-tolerance in peanut cultivars S60,P12,C4,and YS151,including their antioxidant activities,Chl contents,gas exchange,and Chl fluorescence.Shading stress showed greater effects on the peanut yield and biomass accumulation of C4 and YS151,than S60 and P12.Due to the higher activities of SOD,POD and CAT,S60 and P12 had higher ROS scavenging capacities than C4 and YS151 to protect against oxidative stress under long-term shading stress.Under shading stress,S60 and P12 showed higher Chl contents,Pn,Gs,Tr,andFv/Fmthan C4 and YS151,but lower Chla/bratios than C4 and YS151.These results indicate that the light energy absorption and photosynthetic capacities of S60 and P12 were higher than those of C4 and YS151 under shading stress.Furthermore,non-stomatal limitation was the cause of decreased photosynthetic capacity of peanuts under shading stress.S60 and P12 had higher NPQ than C4 and YS151 under shading stress,which helps to dissipate the excess light energy in the form of heat energy and to enhance the plant’s light damage resistance.Meanwhile,peanut yield showed significant correlations with photosynthetic gas-exchange parameters(includingPn,gs,Ci,andTr) and antioxidant enzyme activities(SOD,POD and CAT).Based on yield performance,growth and photosynthetic physiology attributes,S60 and P12 showed higher shading tolerance than C4 and YS151.Our results indicate that these photosynthetic and physiological parameters could be used to evaluate peanut cultivars for shading tolerance.

Acknowledgements

This study was financially supported by the earmarked fund for China Agriculture Research System (CARS-06-135-A17)

Declaration of competing interest

The authors declare that they have no conflict of interest.