Water stress memory in wheat/maize intercropping regulated photosynthetic and antioxidative responses under rainfed conditions

Drought is a most prevalent environmental stress affecting the productivity of rainfed wheat and maize in the semiarid Loess Plateau of China. Sustainable agricultural practices such as intercropping are important for enhancing crop performance in terms of better physiological and biochemical characteristics under drought conditions. Enzymatic and non-enzymatic antioxidant enzyme activities are associated with improved abiotic tolerance in crop plants, however, its molecular mechanism remains obscure. A 2-year field study was conducted to evaluate the influence of intercropping treatment viz. wheat mono-crop (WMC), maize mono-crop (MMC), intercropping maize (IM) and wheat (IW) crops, and nitrogen (N) application rates viz. control and full-dose of N (basal application at 150 and 235 kg ha−1 for wheat and maize, respectively) on chlorophyll fluorescence, gas exchange traits, lipid peroxidation, antioxidative properties and expression patterns of six tolerance genes in both crops under rainfed conditions. As compared with their respective monocropping treatments, IW and IM increased the Fo/Fm by 18.35 and 14.33%, PS-11 efficiency by 7.90 and 13.44%, photosynthesis by 14.31 and 23.97%, C-capacity by 32.05 and 12.92%, and stomatal conductance by 41.40 and 89.95% under without- and with-N application, respectively. The reductions in instantaneous- and intrinsic-water use efficiency and MDA content in the range of 8.76–26.30% were recorded for IW and IM treatments compared with WMC and MMC, respectively. Compared with the WMC and MMC, IW and IM also triggered better antioxidant activities under both N rates. Moreover, we also noted that intercropping and N addition regulated the transcript levels of six genes encoding non-enzymatic antioxidants cycle enzymes. The better performance of intercropping treatments i.e., IW and IM were also associated with improved osmolytes accumulation under rainfed conditions. As compared with control, N addition significantly improved the chlorophyll fluorescence, gas exchange traits, lipid peroxidation, and antioxidant enzyme activities under all intercropping treatments. Our results increase our understanding of the physiological, biochemical, and molecular mechanisms of intercropping-induced water stress tolerance in wheat and maize crops.


Materials and methods
Plant guideline/accordance statement.All the methods included in this study were performed in accordance with the relevant guidelines and regulations.
Plant material.The seeds of local popular wheat and maize cultivars viz.Yongliang 4 and Xianyu 335 were collected from the Northwest A&F University and used with seedling rates of 180 kg ha −1 and 66,670 maize plants ha −1 , respectively.For wheat crop, a 20 cm inter-row spacing both for intercropping and monoculture was maintained.A 50 cm inter-row spacing and 30 cm intra-row spacing, same spacing both for intercropping and monoculture, was kept for maize crop.

Experimentation.
To explore the influence of maize/wheat intercropping on plants' physiological and biochemical events under rainfed conditions, a 2-year field experiment was conducted at Northwest A&F University for two consecutive years (2019 and 2020).The study site has loam soil with > 25% field capacity.During the last four years, the experimental field had been under the cultivation of spring maize crop.Likewise, the study site had the following climatic properties: 14.5 °C and about 500 mm of mean annual temperature and mean annual precipitation, respectively; of which > 70% of rainfall has occurred only between the months of July and September thus crops are frequently exposed the various episodes of water stress (Fig. 1).The soil (above 0-30 cm layer) before the start of the experiment had the following properties: soil pH of 8.12, and 0.8792, 0.0490, 0.0149, and 0.0954 g kg −1 of total nitrogen-N, available N, phosphorus, and potash, respectively.The experimental units were arranged with a randomized complete block design in a split-plot arrangement with three replicates, on the research field of water/moisture stress area.The treatments evaluated were maize monocrop (MM), wheat monocrop (WM), intercropping maize (IM), and wheat (IW) crops under two N application rates: control (without N) and a full dose of N (basal application at 150 and 235 kg N ha −1 for wheat and maize, respectively, for both mono-and intercrops).The total numbers of experimental units were 18.Each experimental unit was 10.5 m in length and 9 m in width and there was 1 m buffer zone between adjacent plots.In relay strip intercropping system, three complete wheat/maize intercropping strips formed a plot.Each strip consisted of eight rows of wheat plants (strip 1.6 m wide) and four rows of maize plants (strip 1.9 m wide).Therefore, 45.7% of the land area in each intercropped plot was occupied by wheat whereas the remaining 54.3% was covered by maize crop.Wheat was sown on October 21, 2019, and October 13, 2020, and maize was sown on April 06, 2020, and March 30, 2021, during the first and second experimental years, respectively.Winter wheat was harvested on June 20, 2020, and 2021, and spring maize was harvested on August 24, 2020, and August 01, 2021.The competitive growth phase between the two crops was about 2 months during both years.Based on the site recommended, phosphorus and potassium were applied at 176 and 40 kg ha −1 by using tricalcium phosphate {Ca 3 (PO 4 ) 2 } and sulphate of potash, respectively.All fertilizers, including treated N, were applied as the basal dose of both crops under both monocropping and intercropping treatments.No irrigation was applied during the experimentation.

Observations and measurements.
Chlorophyll fluorescence and gas exchange.Crops were harvested at the physiological maturity stages of both crops.Young fully expanded leaves of both crops were considered for measuring the chlorophyll fluorescence (CF) and gas exchange attributes by using a portable multifunction photosynthesis system (LI-6400XT; LI-COR, Biosciences, Lincoln, NE, USA).Three plants in each unit were considered for the measurement where data was recorded during the sunny morning hours (8.00-11.30a.m.).Dark-adapted leaves were deemed for the measurement of CF.The highest efficiency of photosystem 2 (PS-II) was revealed through the ratio of variable fluorescence (Fv) to maximum fluorescence (Fm).The ratio of minimum fluorescence (Fo) to Fm was also calculated using the above-mentioned photosynthetic meter.
The measurement for gas exchange attributed included net photosynthesis and stomatal conductance (gs) were made on a portable photosynthesis system where the following settings were fixed: 30 ± 0.01 °C cuvette www.nature.com/scientificreports/temperature, 1.75 cm −2 of fixing leaf area, 495 µm of water flow rate and 40% of relative humidity.Likewise, 396 µmol mol −1 of the surrounding CO 2 cons.and 1500 µmol m −2 s −1 of PAR were also maintained during the measurement.The ratios of photosynthesis to gs and transpiration were considered as intrinsic-(WUEi) and instantaneous-water use efficiency (iWUE), respectively 34,35 .
Leaf free proline.Leaf free proline content was determined following standard procedure.Firstly, 0.2 g of fresh leaf samples of both crops were homogenized in 20 mL solution of aqueous sulphosalicylic acid (SA).Next, 4 mL of the filtered homogenized mixture was blended with 4 mL of acid ninhydrin and 4 mL of glacial acetic acid.After incubating the reaction mixture at a high temperature (100 °C) in a water bath, 8 mL of toluene was then added to the vortexing-reacted mixture.Later, proline contents were estimated from the chromophore, according to the above-mentioned protocol.
Lipid peroxidation.Thiobarbituric acid-based digestion was made for estimating lipid peroxidation in terms of MDA content, as previously described by Heath and Packer 36 .For that, 2 g of fresh leaf samples of both crops were digested in trichloroacetic acid (10%).Next, the digested sample solution was centrifuged at 15,000×g for several minutes at freezing temperature.Later, MDA contents were calculated by considering the supernatant.
Antioxidant enzymes.Total soluble proteins in the leaf tissues were estimated by Bradford 37 method, using fresh leaves samples.The CAT activity was determined following Maehly and Chance 38 .For that, the digestion of fresh leaves samples was made in the H 2 O 2 and phosphate buffer solutions.Later, the absorbance of the reaction mixture was analyzed at 240 nm wavelength, spectrophotometrically considering the molar extinction coefficient of 36 × 103 mM −1 m −1 .Likewise, the pre-described standard protocol of Giannopolitis and Ries 39 , based on p-nitroblue tetrazolium digestion, was followed in order to determine the SOD activity.For that, digestion of fresh leaf samples of both crops was taken place in 10 mL of potassium phosphate buffer solution at chilling temperature.Later, the absorbance was considered at a wavelength of 560 nm at the spectrophotometer, applying the molar extinction coefficient of 4.02 × 103 mol L −1 cm −1 .To determine the POD activity, extraction of fresh leaves sample was made in phosphate buffer solution.Later, the extracted solution was then homogenized in guaiacol.After the addition of H 2 O 2 solution, POD activity was determined spectrophotometrically, according to the standard etiquette of 40 .Furthermore, APX activity in fresh leaf samples was defined according to Yin et al. 41 , where absorbance was made at 290 nm wavelength.
Determination of transcript levels of genes.The transcript levels of the genes related to antioxidant activities including glutathione-S-transferase 1 (GST1), glutathione-S-transferase 2 (GST2), glutathione peroxidase 1 (GPX1), phospholipid hydroperoxide glutathione peroxidase 2 (GPX2), glutathione reductase (GR) and glutathione synthetase (GS) were determined according to the protocol of 42 .Firstly, according to the manufacturer's instructions, total RNA was extracted using the TRIzol reagent.Later, it was treated with RNase-free DNase I (Takara Biotechnology [Dalian] Co., Ltd., Dalian, China) to remove contaminating genomic DNA.About 2 μg of total RNA was used to synthesize the first-strand cDNAs using Super-Script II reverse transcriptase (Invitrogen, Carlsbad, CA, USA).SYBR Premix Ex Taq (Perfect Real Time) kit (Takara Biotechnology [Dalian] Co., Ltd.) on a Light Cycler 480 Real-Time PCR System (Roche Diagnostics Ltd., West Sussex, UK) was used to perform qPCR.The reaction mixture of 20 μL, comprised of 10 μL of SYBR Green Supermix (2×), 1 μL of diluted cDNA, and 0.5 μL of forward and reserve primers, was used.The relative transcript levels were calculated using the 2−ΔΔCt method.The β-actin (GenBank Accession no.AB181991) gene was considered as an internal control.Each data point was expressed as the average ± SD of three independent replicates.

Statistical analysis.
Collected data on physiological and biochemical characteristics were analyzed using Statistix 8.1 software.Two-way ANOVA was applied to evaluate the effects of nitrogen and intercropping treatments.The Tukey HSD test was used to quantify the effects of the treatments i.e., nitrogen and intercropping treatments.Origin-pro software (package 2022) was used to visualize the data graphically.To assess the relationship among tested traits, Pearson correlation analysis was done using the corrplot package.

Results
Chlorophyll fluorescence and gas exchange.Overall water-use efficiency and mean efficiency equivalent ratio.Our results clearly demonstrated that there was a significant difference in water-use efficiency and mean efficiency equivalent ratio among the N treatments, intercropping and experimental years (Table 1).According to our results, there was a significant increase in water-use efficiency and mean efficiency equivalent ratio of intercropping treatments as compared with their respective monocrop treatments.Similarly, N addition (+N) also depicted a significant increase in water-use efficiency and mean efficiency equivalent ratio under all intercropping treatments when compared with control without N application (−N).Moreover, experimental years also affected water-use efficiency and mean efficiency equivalent ratio where there was a significant increase during the second experimental year (Table 1).

Osmolyte accumulation and MDA contents.
Intercropping and N application rates significantly affected the proline, soluble protein, and MDA contents during both study years.Intercropping treatments increased proline and protein contents under both N rates and experimental years except for IW treatment where a slight decrease was noted for proline content during both study years and for N rates (Fig. 4).IM increased the proline content by 18. 75

Antioxidant enzymes.
The activities of all studied antioxidant enzymes varied significantly among the intercropping treatments and N rates.Intercropping and N application significantly (P < 0.001) improved the activities of SOD, CAT, POD, and APX enzymes during both study years (Fig. 5)., respectively in 2019 and 2020, compared with the treatment without N application (Fig. 5).

Person correlation.
The Fo/Fm had a strong positive correlation with photosynthesis, SOD, CAT, and POD, and had a strong negative correlation with MDA.Likewise, PS-II had a strong positive correlation with Gs, WUEi, iWUE, C-Capacity, proline, protein, and APX.Photosynthesis had a strong positive correlation with Gs, protein, SOD, POD, CAT, and APX.GS had a strong positive correlation with WUEi, C-Capacity, proline, protein, and APX, and had a positive correlation with iWUE, SOD, CAT, and POD.WUEi and iWUE had a strong positive correlation with C-Capacity, proline, and with each other, and had a positive correlation with MDA.C-Capacity and proline had a strong positive correlation with protein, APX, and each other.The soluble protein content had a strong positive correlation with antioxidant enzymes and a negative correlation with MDA.Likewise, MDA had a negative correlation with SOD, POD, and CAT.While, SOD, CAT, POD, and APX had a strong positive correlation with each other (Fig. 6).

Transcript level.
In this work, GST1, GST2, GPX1, GPX2, GR, and GS transcript levels were measured using qPCR.The Actin gene was kept as the internal control in both leaf and root tissues of wheat and maize seedlings under different intercropping and N treatments (Fig. 7).The effect of years was nonsignificant for all genes.Intercropping and N treatments significantly affected the relative expression of GST1, GST2, GPX2, and GR whereas there was a non-significant influence on GPX1 and GS both in root and leaves.Among intercropping treatments, intercrops depicted somehow higher expression levels in both root and leaves as compared to monocrops where maize intercropping depicted higher values than the respective monocrop.Among N treatments, the N addition recorded a significantly higher expression level than the control treatment without N addition (Fig. 8).

Discussion
The results supported the hypothesis that intercropping practice is highly effective in improving resource use efficiency and overall crop performance, particularly in rainfed areas.Intercropping treatments significantly improved the chlorophyll fluorescence, and gas exchange traits such as Fo/Fm, PS-II, photosynthesis, stomatal conductance, and C-Capacity when compared with monocropping treatments (Figs. 2, 3).A significant decrease in water use efficiencies (WUEi and iWUE) and MDA contents were noted for intercropping treatments.However, intercropping treatments i.e., IW and IM significantly improved the antioxidant enzyme activities including CAT, SOD, POD, and APX (Fig. 5).Under field conditions, mostly in arid areas, plants endure moisture stress when the required water levels are unobtainable in the rhizosphere, particularly under high evapotranspiration conditions 43,44 .According to previous studies, osmotic stress under moisture stress conditions affected the plant's physiological and biochemical traits 45,46 as has been depicted in this work (Figs. 2, 3, 4, 5, 6).Under extreme dryness, osmotic stress causes stomatal closure, impairs mitosis, and losses of turgor which results in a significant reduction in physiological events including stomatal conductance and photosynthesis 47 as recorded in this study (Fig. 2).Osmotic conditions also disrupt stomatal conductance owing to agitated plant-water relations and reduced activities of photosynthetic pigments and synthesis of phytohormones including ABA that triggers stomatal closure and reduces intercellular CO 2 levels 48 .Nonetheless, intercropping treatments significantly improved the physiological traits of both crops when compared with their respective monocropping treatments under rainfed conditions (Figs. 2, 3).Simultaneously cultivation of C 3 and C 4 crops in the same field alters the ventilation and provides better conditions for light interception 49 .As reported previously, compared with C 3 crops, C 4 had more height and showed higher light saturation levels for better photosynthesis 50,51 .Moreover, previous studies have demonstrated that the combination of C 3 and C 4 , as high-position crops, provided compensation while capturing the sunlight for better photosynthesis 52,53 .It is generally claimed that chlorophyll fluorescence is an important indicator to draw the relationships between the photosynthetic levels and the www.nature.com/scientificreports/surrounding environments; this phenomenon is critical in estimating the efficacy of intercepted light, and the absorption and distribution of captured light during photosynthesis 54 .Estimating the efficacy of PSII and Fo/ Fm is critical to determine the plants' photosynthetic efficiency 55 .In this work, our results demonstrated that intercropping treatments significantly improved the efficacy of PSII, Fo/Fm, and overall photosynthesis when compared with respective monocropping treatments.Higher photosynthesis under intercropping might be associated with better activities of photosynthesis enzymes, as previously reported by 51 .In C 4 plants, PEPC and RuBisCO enzymes play essential roles during the process of carbon assimilation, in this way can determine the efficacy of leaf photosynthesis 51 .According to previous reports, RuBisCO plays critical roles in carboxylation and oxygenation during photosynthesis; this enzyme is a key driver of photorespiration 56,57 .Furthermore, it is also well demonstrated that the PEPC enzyme is involved in the fixation of primary carbon dioxide in C 4 plants 58 .
Under moisture stress, plants also experienced numerous changes at biochemical levels including the excessive production of ROS that results in lipid peroxidation and membrane damage 59 .Malondialdehyde, a product of lipid peroxidation, is demonstrated responsiveness to oxidative stress 60 .Consequently, malondialdehyde contents are used as an indicator of plant tolerance to abiotic stresses 61 .In this work, intercropping treatments depicted significantly lower values of MDA content showing higher tolerance to water deficit stress.Furthermore, as an adaptive response, numerous antioxidative enzymes such as SOD, POD, CAT, and APX are also produced in plants to detoxify the ROS-induced effects 62,63 .In this work, our results demonstrated that intercropping treatments significantly increased the activities of antioxidant enzymes as compared with their respective monocropping treatments (Fig. 4).Similar to our results, Zheng et al. 51 reported that intercropping treatments substantially increased the activities of SOD, POD, CAT, and APX enzymes and decreased lipid peroxidation by decreasing the MDA activities.Recently, Cui et al. 64 studied the influence of intercropping treatments on antioxidant enzyme activities and reported that these treatments decreased the toxic effects of oxidative stress by increasing the activities of antioxidant enzymes.Antioxidative enzymes such as CAT, SOD, APX, and POD, are proficient to sustain ROS levels and counteract the plants from lipid peroxidation (membrane damage).Indeed, under high ROS conditions, plants can protect cells from oxidative stress by inducing a strong antioxidative defense system 65,66 .Previously, Cho and Seo 67 proposed that antioxidant enzymes sustain and modulate the H 2 O 2 levels for signaling during metabolic alterations under stressful conditions.In this work, IM and IW treatments significantly improved the activities of antioxidants in both maize and wheat crops, compared with monocropping treatments.Moreover, antioxidant enzymes had a negative correlation with the MDA content, showing that IM GSH and ASA are major non-enzymatic antioxidants that play significant roles in the scavenging of ROS 68 .Overexpression of the genes related to non-enzymatic antioxidants confers enhanced tolerance to abiotic stresses in crop plants 69 .Transcriptional analysis helps to quantify the changes in transcript levels of genes that are involved in the regulation of metabolism.In this work, the expression levels of six genes encoding ASA-GSH cycle enzymes were determined in wheat-maize seedlings exposed to various intercropping treatments and N application.According to our results, in root and leaf tissues of both wheat and maize seedlings under intercropping treatments and N rates, the transcript profiles of ASA-GSH synthesis-related genes varied and intercropping treatment markedly enhanced the expression of these genes.In line with these results, Li et al. 70 also reported enhanced expression levels of various genes involved in regulating the activities of enzymatic and non-enzymatic antioxidants.Similar was also reported by Wei et al. 71 who identified various genes regulating the activities of non-enzymatic antioxidants.
Under water stress, to maintain cellular hydration, through osmotic adjustment, crop plants also accumulate solutes that work as osmolytes and play critical roles in the protection of cellular structure 72,73 .It has been reported that intercropped crops can accumulate more solutes for their survival under moisture stress than monocrops 74 .Similar was found in this work in which intercropping treatments significantly improved proline and protein contents in both crops (Fig. 5).Furthermore, a strong positive correlation among proline, protein, and antioxidant enzymes also showed that these enzymes and solutes accumulation help to sustain intercropping crops under moisture stress conditions (Fig. 6).The synthesis and accumulation of proline also take place in plants to induce tolerance to water stress 75 .It has been well established that proline also owns antioxidative belongings and protects the plant cells from dehydration when acting as chaperones to shield the macromolecule assembling 76 .The radical scavenger properties of proline are also well demonstrated in previous studies 77,78 .

Conclusion
Our results indicated that both maize and wheat crops when grown under intercropping system performed better under rainfed conditions than their respective monocrops.Intercropping treatments with significantly higher proline and protein contents, better chlorophyll fluorescence, the activities of antioxidative enzymes such as SOD, POD, CAT, and SOD, and lower MDA levels were better able to endure their growth and development under moisture deficit conditions.Use of these traits i.e., chlorophyll fluorescence, antioxidative enzyme activities, and osmolytes accumulation will be of interest in future breeding programs to produce drought-tolerant genotypes, particularly for rainfed conditions.

Figure 1 .
Figure 1.Daily weather data including the precipitation and average temperature during both experimental years.

Figure 2 .
Figure 2. Fo/Fm, PS-II efficiency, photosynthesis, and stomatal conductance (gs) under different intercropping treatments (maize monocrop (MMC), wheat monocrop (WMC), intercropping maize (IM), and wheat (IW) crops) and nitrogen treatments in 2019 and 2020.Different lower-case letters show a significant difference among intercropping treatments under both N rates."*" above bars indicates a significant difference between the monocropping and intercropping treatments at *P < 0.05; **P < 0.01.Note: Fo and Fm indicate the minimum and maximum chlorophyll fluorescence in the dark-adapted state, respectively.

Figure 6 .
Figure 6.Pearson correlation coefficient of chlorophyll fluorescence, gas exchange attributes and antioxidants under different intercropping treatments and N application rates (n = 3).Fo and Fm indicate the minimum and maximum chlorophyll fluorescence in the dark-adapted state, respectively; gs donates stomatal conductance; WUEi and iWUE indicate the intrinsic and instantaneous water use efficiency, respectively; MDA shows malondialdehyde; CAT, SOD, POD and APX indicate catalase, superoxide dismutase, peroxidase, and ascorbate peroxidase activity, respectively.

Figure 7 .Figure 8 .
Figure 7. Effects of intercropping treatments and N rates on transcript levels of the six genes encoding ASA-GSH cycle enzymes in root of both wheat and maize crops during both years.Transcripts were analyzed by qPCR using Actin gene as internal control.The three seedlings were collected in one replication and three independent biological replications were performed.Each value is the mean ± standard error of three independent measurements.Intercropping treatments were: (maize monocrop (MM), wheat monocrop (WM), intercropping maize (IM), and wheat (IW) crops).Different lower-case letters show a significant difference among intercropping treatments under both N rates.

Table 1 .
Effect of wheat-maize intercropping system on water use efficiency and mean water use efficiency equivalent ratio during both study years under with-(+ N) and with-out nitrogen (−N) application.MMC, Maize monocrop; WMC, wheat monocrop; IM, intercropping maize; IW, intercropped wheat.