Introduction

The atmospheric CO2 concentration has been increasing at an alarming rate, since the onset of industrial revolution. It was approximately 280 parts per million (ppm) during the pre-industrial period, has now reached around ~ 410 ppm, and it is projected to continue rising in the future1,2,3. Based on the current emission rates, it is projected that CO2 levels might increase to 550 ppm by 20502. Climate models indicate that Earth’s near-surface temperature may also experience a significant rise of 1.4 to 5.8 °C as a result of higher concentrations of CO2 and other greenhouse gases4,5,6. These predictions underline the urgency of addressing this issue and taking measures to mitigate the impact of climate change.

The elevated atmospheric CO2 level is generally known to have a positive effect on net photosynthesis rate, growth, and yield of crops7,8,9. Research has shown that an increase in atmospheric CO2 can benefit plant biomass in cereals like barley, wheat, rice, oat, and rye by enhancing the net photosynthesis rate10,11,12. The free-air carbon dioxide enrichment (FACE) studies at eCO2 levels also revealed increased photosynthetic rate by almost 40% in different plant species13,14. A comprehensive analysis of 186 independent studies conducted on eighteen C3 crops revealed that an elevation of CO2 levels led to an average increase in yield by 18%15 under non-stress conditions. These findings provide strong evidence for the positive impact of eCO2 on crop productivity, indicating that higher concentrations of CO2 in the atmosphere can enhance the growth and yield of these crops. The positive effect of elevated CO2 (eCO2) is more evident in C3 crops compared to C4 crops such as maize16. The C4 plants, with their efficient CO2 fixation process and carbon concentrating mechanism in bundle sheath cells17,18, generally show less stimulation of photosynthesis and growth under eCO2 compared to C3 plants. This efficient mechanism allows C4 plants to minimize stomatal conductance, leading to greater drought tolerance compared to C3 species19,20. The increase in biomass under eCO2 can be attributed to enhanced carbon fixation and an extended active growth21 and grain filling period leading to higher crop yields. Additional evidence demonstrating the benefits of eCO2, in terms of increased photosynthetic rates, foliar C/N ratio, enhanced plant growth and yield which has also been reported in potato, lettuce, sunflower, barley, and wheat22,23,24,25,26.

The increasing CO2 levels are also the reason for an increase in ambient temperatures, which influences the crop phenology and duration. The elevated temperature (eT) leads to shorter crop life cycle by reducing the duration of different phenophases in rice, wheat, maize and mung bean27,28,29. The prolonged exposure to high temperatures has been reported to increase photo-respiration in plants30 and, also affect the photosystem II activity, chlorophyll concentration, and enzyme activities31. The elevated temperature also negatively influences the photosynthesis in addition to heat injury, and metabolic disorders resulting reduced yield32. The maize yield in Corn Belt region of US was predicted to reduce by 8% with every 2 °C increase in temperature33. In case of wheat production in India, temperature rise of 0.5–1.5 °C decrease the yield potential from 2 to 5%34, while an increase by 1 °C is projected to reduce overall production by 4 to 5 million tonnes35. An increase in mean canopy temperature by 2.7 °C throughout the crop growing season decreased seed yield in maize, regardless of ambient or elevated CO236. In a rice FACE system, elevated CO2 increased grain yield by 14%, but there was no significant temperature effect at the relatively cool site37. The optimal temperature range for the growth and productivity of maize crops is typically between 22 and 32 °C, with a minimum range of 16.7–23.3 °C. However, extreme temperatures can significantly impact maize productivity. When temperatures drop below 5 °C or rises above 32 °C, it can have adverse effects on the yield of maize crop38. Therefore, maintaining temperatures within this suitable range is crucial for maximizing maize productivity. The temperature higher than 32 °C during anthesis silking interval (ASI) in maize drastically affects seed setting. Elevated atmospheric CO2 levels can mitigate the adverse effects of moisture deficit stress on maize by reducing stomatal conductance and water loss, while simultaneously preserving soil moisture and maintaining optimal yield39.

The studies reveal that eCO2 concentration increases the productivity of C3 (15–41%) and C4 (5–10%)40. The response of maize crop to elevated CO2 indicated a stimulation of biomass by 3–6%41, with some reports suggesting an increase of even up to 50%42. Elevated CO2 enhances maize plant height, leaf area and above ground biomass, resulting in an improved yield and yield related components43. The experiments with OTCs showed that eCO2 at 550 ppm increased the biomass and grain yield of maize crop44. Elevated CO2 along with elevated temperature increased stover yield, grain yield and harvest index (HI) of maize compared to ambient CO2. The elevated CO2 ameliorated the negative impacts of elevated temperature on yield and yield components of maize28. These studies revealed that C4 maize crop have the potential to respond positively to elevated CO2 like C3 crops. Majority of the studies reported on individual effects of elevated carbon dioxide and temperature on phenology, physiology and biochemistry of different crops45,46,47,48,49. Although the combined and interactive effects of elevated CO2 and temperature on physiology, phytochemistry, and biomass have been attempted in limited manner36,50,51,52. There is very few research studies to investigate these interactive effects on C4 crops, specifically with maize and also genotypic variability. It is important to understand the interaction of these climate variables on the growth and yield of crop plants, as changes in CO2 concentration and temperature are likely to occur concurrently53. With this background, the present study was aimed to assess the responses of popular maize hybrids under elevated temperature (eT) and its interaction with elevated CO2 (eT + eCO2) conditions, focusing on phenology, physiology, biochemical, biomass and grain yield traits.

Materials and methods

Plant material and growth conditions

A field study was conducted under Free Air Temperature Elevation (FATE) facility during rainy season for two years, at Central Research Institute for Dryland Agriculture (ICAR-CRIDA) Hyderabad, Telangana, India. The geographical coordinates of the research site are approximately 17.20° N latitude and 78.30° E longitude. The study was to assess the growth and yield responses of four popular maize hybrids (DHM117, DHM121, NK6240, and 900M GOLD) at elevated levels of atmospheric carbon dioxide (eCO2) and increased temperature (eT). Among the selected maize hybrids, the 900M GOLD hybrid, developed by Bayer Crop Sciences, stands out for its high-yielding potential. This particular hybrid is well-suited for different regions in India, making it a favourable choice for cultivation across states. Another widely cultivated hybrid is the NK6240, developed by Syngenta Pvt. Ltd. This hybrid has gained recognition for its exceptional adaptability, consistently delivering stable yields in diverse environmental conditions. Additionally, the study included two locally developed hybrids, DHM117 and DHM121 from Telangana State Agricultural University. These hybrids were specifically bred to thrive in the rainfed conditions prevalent in Telangana State.

The FATE facility consisted of nine rings, each with a diameter of 8 m, enabling the maintenance of three treatment conditions. This design allowed for controlled experimentation and the examination of the effects of elevated temperature and CO2 on maize hybrids within the specific planting configuration. The crop was sown with a spacing of 0.30 m between plants and 0.75 m between rows. It's worth noting that the plant density within the FATE rings was intentionally set different from the recommended spacing of 0.25 m (plant to plant) and 0.60 m (row to row), typically followed in rainfed agro-ecologies. This specific spacing was chosen to ensure that each plant is exposed to elevated levels of temperature and CO2 released by the infrared heater and CO2 system within the ring.

The study consisted of three distinct treatment levels. The first level served as the control treatment, maintaining ambient conditions with a CO2 concentration of ~ 400 ppm and the temperature at ambient levels (aT). The second level involved an elevated temperature (eT), with the temperature set at ambient levels + 3 °C ± 0.5 °C. The third treatment level combined elevated temperature and CO2 (eT + eCO2), simulating environmental conditions with both the temperature set at ambient levels + 3 °C ± 0.5 °C and elevated CO2 level of 550 ± 50 ppm. Three FATE rings having similar treatment conditions were used as three replications (Plate 1). Each ring, with a total area of 50.26 m2, was further divided into four equal quadrants, with each quadrant having an area of 12.56 m2. This division was done to allocate space for sowing each hybrid in each quadrant, ensuring a randomized distribution of the hybrids across the different rings. By implementing this design, the study aimed to minimize any potential bias that could arise from the placement of the hybrids within the experimental setup.

Plate 1
figure 1

Experiment under FATE facility.

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To maintain elevated crop canopy temperature (eT) of ambient + 3 °C ± 0.5 °C, each warming ring was fitted with 24 ceramic infrared heaters assembly consisting each of 2 × 1000 W capacity (Elstein, model FSR-1000W) above the canopy. The heating system provides warming without any photo-morphogenic effects or significant radiation emitted below 850 nm wavelengths. Out of six warming rings, three rings were also provided with CO2 release system at 0.30 and 0.8 m height from the base of the ring to assess the interactive effects of elevated temperature and CO2. The polyurethane (PU) tubing along the fringe of the ring with laser drilled perforations releases CO2 within ring to maintain the elevated concentration of 550 ppm. The CO2 release was controlled by solenoid valves, which in turn was regulated by the SCADA based control system linked with CO2 analyser, wind direction and wind speed. The CO2 concentration at the centre of the ring was continuously monitored by IRGA based CO2 analyser (Priva, model-200821) and the duration of CO2 release was based on the set CO2 concentration for the specified area as well as wind direction and wind speed. The canopy temperatures were monitored with infrared sensor (Ray teck Fluke, model-RAYCMLTJ3) fitted in each ring. The duration and intensity of heating of warming plots is regulated by proportional-integral-derivative (PID) system using canopy temperatures of control plots as reference11. The set conditions were maintained for 24 h throughout the crop season, starting from the germination and continued until the physiological maturity of the crop. The signals from each sensor are being monitored, recorded and controlled by Program Logic Control (PLC) and Supervisory Control and Data acquisition (SCADA) system. The recommended doses of fertilizers were applied in three splits @ 60 kg N ha−1 and 60 kg P ha−1, 30 kg K ha−1 as muriate of potash as basal; 30 kg N ha−1 as second dose at knee- high stage and 30 kg N ha−1 and 30 kg potassium ha−1 as third dose was applied at tasselling stage. The crop was raised with supplemental irrigation at regular intervals and maintained at optimum soil moisture along with recommended plant protection measures to control pests and diseases.

Weather during crop growth period

The weather parameters of two seasons during crop growth period were presented in Fig. 1. In season one, the maximum air temperature during vegetative stage of the crop ranged from 25.6 to 35.6 °C with an average value of 30.84 °C while minimum temperature ranged from 20.0 to 25.7 °C with an average value of 21.9 °C. While, during grain filling stage, maximum temperature ranged from 24.0 to 33.6 °C with an average value of 29.47 °C and minimum temperature from 18.8 to 23.4 °C with an average value of 21.1 °C. In season two, during vegetative stage of the crop, the maximum air temperature ranged from 23.4 to 35.0 °C with an average value of 31.9 °C while minimum temperature ranged from 21.0 to 25.0 °C with an average value of 23.1 °C. During grain filling stage, maximum temperature ranged from 24.2 to 34.6 °C with an average value of 31.4 °C and minimum temperature from 19 to 24 °C with an average value of 22.7 °C. The maximum humidity in first season varied between 61 to 100%, while minimum was from 38 to 98%. In the first season crop received 700 mm rainfall. In case of second season, maximum humidity varied between 79 to 98%, while minimum was from 33 to 93% and crop received a total of 770 mm rainfall.

Figure 1
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Weather parameters of two crop seasons where the experiments was conducted.

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Observations recorded

Phenological parameters

The phenological observations of days to 50% tasselling, anthesis and silking were recorded when 50% of the plants had tassel initiated, pollen shed and had emerged silks respectively. The anthesis silking interval (ASI) was calculated as number of days between days to anthesis and days to silking.

Physiological parameters

All physiological parameters were recorded on the fully expanded third leaf from the top. The relative water content (RWC) was determined following standard protocol54 i.e. RWC (%) = [(FW − DW)/(TW − DW)] × 100, where FW—fresh leaf weight, TW—turgid leaf weight after rehydration, DW—the dry leaf weight after oven drying.

The net photosynthetic rate (Anet), stomatal conductance (gs), transpiration rate (Tr), internal CO2 concentration (Ci), leaf temperature (Tleaf) and vapour pressure deficit (VPD) were measured during anthesis stage. The measurement was taken on fully expanded third leaf from uppermost active leaf in three representative plants for each hybrid. The measurements were recorded between 10:00 and 12:00 h using portable photosynthesis system (LI-6400, LI-COR, Nebraska, USA) with irradiance set at 1200 µmol m−2 s−1 under three conditions viz., aT, eT, and eT + eCO2. The conditions of temperature and CO2 in the leaf chamber of IRGA were set as that of the plot conditions. The water use efficiency (WUE) was calculated as the ratio of Anet and Tr using the formula WUE = Anet/Tr.

Biochemical parameters

To estimate alcohol soluble metabolites, fresh leaf tissue of 0.5 g was homogenized in 80% ethanol by grinding using mortar and pestle. The homogenized sample was centrifuged at 24 °C, 10,000 rpm for 20 min and the supernatant was stored in screwcap tubes in refrigerator. The supernatant was used for estimation of total soluble sugars, free amino acids and starch content.

Total soluble sugars were estimated by the sulphuric acid-phenol method55, where 0.1 mL of supernatant was taken in a test tube and 1 mL of phenol reagent was added followed by 5 mL of concentrated sulphuric acid and mixed thoroughly. The samples were incubated for 30 min at room temperature and after the colour development absorbance was measured at 490 nm by using spectrophotometer and expressed as mg g−1 fresh leaf weight.

The free amino acids content was determined by using method of Moore and Stein56, where 0.1 mL of supernatant was taken in test tube and mixed with 1 mL of freshly prepared ninhydrin reagent and volume was made up to 2 mL with distilled water and then heated in boiling water bath for 20 min. After cooling, 5 mL of propanol was added to the above mixture and kept for 15 min. The absorbance was read at 570 nm by using spectrophotometer and expressed as mg g−1 fresh leaf weight.

Starch content was determined by anthrone method57. The residue (pellet) was washed repeatedly with 80% ethanol till the washing does not give colour with anthrone reagent and then dried. Water and perchloric acid (52%) at the ratio of 1:1 was added into pellet and centrifuged. The process was repeated twice and obtained supernatant was used for measurement of starch content. The absorbance was read at 630 nm by using spectrophotometer and expressed as mg g−1 fresh leaf weight.

The lipid peroxidation was estimated in terms of malondialdehyde (MDA) content following Health and Packer58 method. To estimate MDA content, 1.0 g of leaf tissue was grinded in 2.0 mL of TBA and centrifuged at 10,000 rpm for 10 min at 4 °C. In a test tube two mL of supernatant and 4 mL of 0.5% TBA were added. The test tubes covered with aluminium foil were heated at 95 °C for 1 h and immediately cooled in ice bath. The mixture was centrifuged for 5 min at 10,000 rpm. Then the supernatant was collected and the absorbance was read at 532 and 600 nm by using spectrophotometer to measure the MDA content and expressed as μmol/g FW.

Proline content was estimated following Bates59 method. One gram of fresh leaf tissue was homogenized in 3% aqueous sulphosalicylic acid using mortar and pestle and centrifuged at 10,000 rpm at 24 °C for 10 min. Acid-ninhydrin solution (1.25 g ninhydrin in 30 mL glacial acetic acid) was added and heated at 90 °C for 1 h. The reaction was terminated by placing the tubes in ice bath, and then extracted with 4 mL of toluene by vortexing for 1 min. The absorbance was read at 520 nm in spectrophotometer using toluene as a blank and expressed as μM proline/g of fresh leaf weight.

Biomass and yield parameters

At the maturity, three plants of each hybrid from every replication under three different conditions (aT, eT and eT + eCO2) were carefully uprooted. These plants were then separated into their respective components, including leaves, stems, roots, and cobs. To ensure accurate measurements, the roots were thoroughly washed to remove any soil particles. Subsequently, the harvested plant parts were subjected to a drying process in a hot air oven set at 55 °C. The drying continued until the plant samples reached a constant weight for determination of dry weights. The dry weight of leaf, stem, and root was measured using scientific balance and expressed as gram per plant. The yield parameters—cob weight (g/plant), seed number, seed yield (g/plant), test weight (hundred seed weight), total biomass, vegetative biomass, and harvest index (HI) was recorded. The entire experimental setup was conducted under controlled conditions with predefined parameters and hybrid, being one hundred percent heterozygous and homogeneous in nature, the average of three plants data per replication represent its actual responses to different treatments. Harvest index was calculated as \(HI=\left(\frac{Grain\, yield}{Total\, biomass}\right)\times 100\) and expressed in percentage.

Statistical analysis

The replicated data of individual season and combined over seasons were subjected to statistical analysis following randomized complete block design (RCBD) using SAS software ver. 9.3 to assess the significance of treatments, hybrids and their interaction. The analysis of variance was applied to compare hybrids in individual trial and combined over treatments. Subsequently, the Tukey’s Honest Significant Difference (Tukey’s HSD) test was used post-hoc to identify the significant treatments and hybrids. All statistical tests were conducted at 5% level of significance. The R statistical programming language was used to visualize the results from ANOVA and Tukey’s HSD test.

Ethics declarations

All plant experiments were conducted in accordance to relevant institutional, national, and international guidelines and legislation.

Results

The combined analysis of variance (ANOVA) revealed significant variances for most of the traits related to phenological, physiological, biochemical, biomass and yield traits due to hybrids, treatments and treatments × hybrids interaction (Table 1).

Table 1 Combined analysis of variance (ANOVA) for physiological, biochemical and yield related parameters.
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Phenology

There was differential responses of anthesis and silking among hybrids under elevated temperature (eT) which influenced the crop phenology, specially affecting the days to anthesis and silking (Table 2) resulting increase in ASI as compared to aT. However, under eT + eCO2 conditions, presence of eCO2 partially mitigated the negative effects of eT (Fig. 2A). Among hybrids tested, DHM117 had the highest ASI (4.33 days), followed by DHM121 (3.67 days), NK6240 (3.17 days) and 900M GOLD (2.5 days) under eT conditions (Table 3).

Table 2 Means of phenology and biomass parameters at different treatment level.
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Figure 2
figure 3

Performance of maize hybrids under different treatment conditions for crop phenology and yield related traits. Data are given as mean ± SD. Treatments with different grouping letters are significantly different.

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Table 3 Means of phenology and biomass parameters at treatment × hybrids level.
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Biomass

There were significant differences between hybrids under elevated temperature for total biomass, and its vegetative and yield components (Table 2). Among the hybrids, the reduction of leaf biomass at eT varied from 5.5% (900M GOLD) to 15% (NK 6240) and total biomass from 5.44% (900M GOLD) to 13.3% (DHM117). Notably, the hybrid DHM117 exhibited better recovery in leaf, shoot, and total biomass under eT + eCO2 condition, reaching levels comparable to the ambient treatment (Table 3). Under ambient condition, the total biomass was highest and similar across all hybrids, followed by eT + eCO2 and eT. Additionally, the hybrid 900M GOLD recorded significantly higher biomass under eT as compared to other hybrids, whereas, DHM117 and DHM121 demonstrated the ability to recover under eT + eCO2 condition (Fig. 2B).

Yield parameters

The combined analysis of variance (ANOVA) revealed highly significant (p < 0.01) variances for cob weight, seed number, seed yield, and hundred seed weight due to hybrids, treatments and their interaction. The impact of elevated temperature was found to be significant in reducing the yield components however, the magnitude of response varied with individual hybrid (Table 4). Specifically, NK6240, DHM117, and DHM121 experienced reduction in cob weight by 11.61%, 14.11% and 13.41% respectively (Fig. 2C). The most significant reduction in seed yield (19.32%) was observed with DHM117 (Fig. 2D) which is primarily attributed due to a decrease in seed number (12.67%). Among the four maize hybrids, 900M GOLD had higher cob weight, seed number, and hundred seed weight as it responded positively for yield components under both eT and eT + eCO2 conditions (Table 5). It demonstrated the least reduction in cob weight (4.23%), seed yield (2.06%) under eT conditions, also showing an increase in hundred seed weight (5.13%) indicating its tolerance to high temperature.

Table 4 Means of grain yield related traits at different treatment levels.
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Table 5 Means of grain yield parameters at treatment × hybrids level.
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Physiological parameters

The ANOVA revealed significant variances attributed to the treatment, treatment × hybrids interaction (p < 0.01) for Anet, gs, Tr, leaf temperature, internal CO2 and vapour pressure deficit. The crops grown under eT condition had significantly lower values of Anet, gs and Tr compared to those under aT condition (Fig. 3A–C). The Anet was particularly affected by eT, the crop showed some degree of recovery under eT + eCO2 (Table 6). Both transpiration rate and stomatal conductance also decreased significantly under eT. Leaf temperature was significantly lower under aT as compared to eT and eT + eCO2 while internal CO2 content was lowest under eT followed by aT and eT + eCO2. The VPD was significantly higher under eT as compared to aT, followed by eCO2 + eT. Among the hybrids, 900M GOLD maintained relatively higher Anet, gs, and Tr at eT, indicating its ability to capture more CO2. The Anet of all hybrids reduced under eT but, the presence of eCO2 facilitated its recovery in DHM117 and 900M GOLD, reaching levels similar to those in the ambient plots. DHM121 and NK6240 showed > 98% recovery under eT + eCO2 condition. Among the maize hybrids, 900M GOLD consistently maintained highest Anet under all the three conditions, with the lowest impact of eT. Although DHM117 experienced the greatest reduction of Anet (28.4%) under eT, it was able to recover to that ambient levels in the presence of eCO2 (Table 7). The leaf temperature increased significantly in hybrids under eT and eT + eCO2 as comparison to aT. In contrast, internal CO2 decreased under eT but showed a significant increase under eT + eCO2 conditions. However, the response of genotypes varied, particularly in the case of DHM117, where internal carbon significantly decreased under eT, possibly due to stomatal closure, leading to a reduced Anet. Conversely, DHM121 exhibited higher internal CO2 under eT as compared to aT, aligning with a lesser impact on Anet and stomatal conductance (gs), and also maintaining better RWC resulting in the higher accumulation of CO2. Furthermore, the vapor pressure deficit (VPD) among hybrids was markedly higher under eT compared to aT and showed a reduction in the presence of eCO2 under the eT + eCO2 conditions.

Figure 3
figure 4

Performance of maize hybrids under different treatment conditions for physiological traits. Data are given as mean ± SD. Treatments with different grouping letters are significantly different.

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Table 6 Means of physiological and biochemical parameters at different treatment level.
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Table 7 Means of physiological and biochemical parameters at treatment × hybrids level.
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The reduction in gs due to elevated temperature varied across hybrids, ranging from 5.56% (DHM121) to 47.62% (DHM117). Similarly, the reduction in transpiration rate ranged from 2.80% (DHM121) to 36.76% (DHM117) under eT condition. Under eT + eCO2 condition, reduction in gs ranged from 7.14% (DHM117) to 31.11% (900M GOLD) and for Tr it varied from 6.98% (DHM117) to 16.53% (900M GOLD). Notably, DHM117 had maximum recovery (> 90%) in gs and Tr under eT + eCO2 condition, while a linear reduction was observed for 900M GOLD under both eT and eT + eCO2 conditions. DHM121 showed higher reduction in gs and Tr under eT but displayed greater recovery under eT + eCO2 condition. In DHM117, the reduction in Tr due to eT was higher than the reduction in Anet, resulting in higher water use efficiency (WUE) compared to ambient condition. All hybrids recorded higher WUE under eT + eCO2 condition, where the presence of elevated CO2 helped in the recovery of Anet even with eT, leading to increased WUE (Fig. 3D). The impact of eCO2 was more prominent in reducing Tr of DHM121 and NK6240, while it improved Anet with DHM117 and 900M GOLD (Table 7).

To assess the leaf water status of plants, the relative water content (RWC) was estimated under different treatments (Fig. 4A). Under ambient condition, the RWC was approximately 87%, indicating good cellular hydration. However, eT caused a significant reduction in RWC, dropping to 78%. Interestingly, under the eT + eCO2, the RWC recovered to around 83%, demonstrating the positive effect of eCO2 in maintaining better leaf water status even under high temperature (Table 6). Among hybrids, 900M GOLD exhibited the ability to maintain a significantly higher RWC (82%) under eT compared to the other hybrids, while DHM117 was most affected (75%). However, DHM117 recovered substantially under eT + eCO2 (Table 7). Overall, the negative impact of eT was mitigated to some extent under eT + eCO2 treatment for all hybrids.

Figure 4
figure 5

Performance of maize hybrids under different treatment conditions for physio-biochemical traits. Data are given as mean ± SD. Treatments with different grouping letters are significantly different.

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The level of lipid peroxidation, indicated by measurement of MDA content, exhibited a significant increase under the eT condition compared to ambient control (Table 6). Among hybrids, DHM117 showed the highest MDA content (20.47 mg/g fresh weight) under eT while 900M GOLD had the lowest level (15.99 mg/g fresh weight) (Fig. 4B). However, under eT + eCO2 condition the impact of eT was mitigated, resulting in lower MDA content.

There was significantly higher accumulation of proline in all the hybrids under eT condition (Fig. 4C). The accumulation of proline was more than double in DHM117 under eT as compared to ambient control. However, the impact eT was mitigated under eT + eCO2, indicating the beneficial role of eCO2. Among the hybrids 900M GOLD was found to be more stable under different treatments in terms of proline accumulation (Table 7).

The hybrids had significantly lower free amino acid (FAA) compared to ambient condition (Table 6) under eT and eT + eCO2, although there was variation among hybrids. Among hybrids, 900M GOLD, DHM121 and NK6240 maintained similar FAA content under eT and eT + eCO2 conditions, while DHM117 showed reduced FAA accumulation. The eT and eT + eCO2 conditions led to increased TSS accumulation, with highest accumulation observed in DHM117. In contrast, 900M GOLD showed lowest TSS accumulation under eT, indicating that the metabolic activities of this hybrid were less affected under eT. The starch accumulation under different treatments did not vary much among hybrids under different treatments, although relatively higher starch (Fig. 4D) accumulation was observed under eT + eCO2 condition compared to ambient and eT (Table 7). Plants tend to remobilize more starch under elevated temperature and CO2 to provide energy and carbon when photosynthesis is potentially limited. The release of sugars and other derived metabolites support plant growth under stress and function as an osmo-protectants to mitigate the negative effect of stress. The carbon content did not show much variation among hybrids under different treatments, although relatively higher carbon accumulation was observed under eT + eCO2 condition compared to ambient and eT conditions.

Discussion

A meta-analysis examining the impact of climate change on plants revealed clear evidence that physiological, growth, and yield related traits were influenced by eT and eCO260,61. It is evident that crops respond to changing climatic conditions through intricate phenological, physiological, and biochemical processes. The primary objective of the present study was to assess the impact of elevated temperature individually and in combination with elevated CO2 (eT + eCO2) on maize, a C4 crop. Additionally, the study sought to quantify the role of elevated CO2 in mitigating the adverse impacts of elevated temperature on maize plants. By examining the interactive effects of these factors, the research aimed to provide an insight into the potential benefits of eCO2 in alleviating the ill effects of elevated temperature, specifically in the context of maize cultivation. The availability of diverse maize hybrids in the seed chain has also made it possible to estimate the variability among hybrids cultivated by farming communities under various production systems.

The intricate interplay between abiotic stresses, notably drought and elevated temperature, has been recognized for its discernible impact on the temporal dynamics of maize flowering, specifically influencing the tasselling, anthesis and silking in maize62. In our study, we investigated the ramifications of eT and the synergistic interplay of eT + eCO2 on phenology of flowering of maize. Specifically, eT accelerated the onset of anthesis and silking. This acceleration, however, was not replicated under the combined influence of elevated temperature and carbon dioxide (eT + eCO2), which intriguingly manifested behavior akin to ambient conditions. The ASI increased under elevated temperature (eT) conditions due to the early onset of anthesis compared to the requisite days for silk emergence. The variable responses exhibited by different hybrids under these conditions underscored their inherent genetic potential.

Among the hybrids, DHM117 exhibited a higher ASI under the set levels of elevated temperature condition, indicating its sensitivity to high temperature. In contrast, 900M GOLD displayed a relatively lower ASI, suggesting a greater tolerance or resilience to the set level of elevated temperature. The ASI is a critical trait in maize crop for ensuring successful fertilization and proper seed setting. These divergent responses of these hybrids to elevated temperature offer valuable insights into their adaptative capacities and performance under challenging environmental conditions. Aligning with existing research, our findings echo the trend of elevated temperature in reducing the days to anthesis or silking in maize hybrids63. In another study, it was observed that warmer temperature mainly affected the reproductive stages and thereby grain yield was significantly reduced to 80–90% as compared to normal condition64. In the present study, we made an interesting observation that the ASI of sensitive maize hybrids (DHM117 and DHM121) showed some degree of recovery under eT + CO2 condition. This finding highlights the potential ameliorative effect of elevated CO2 on phenological parameters. As climate change continues to exert its complex influence, the intricate relation between temperature, CO2, and maize crop invites for further exploration into the adaptive mechanisms that may shape agricultural resilience.

Generally, plants with different photosynthetic pathways exhibit a complex distinct response to eCO2 and temperature. The C3 crop species, known for their increased photosynthesis under eCO2 conditions, stand in stark contrast to the C4 plants, which, due to their efficient CO2 concentrating mechanism, exhibit a more modest enhancement in net photosynthesis and biomass. In the present study, Anet of maize hybrids was reduced at eT and, however the magnitude of response of individual hybrid varied. Among the hybrids, 900M GOLD recorded the highest per se values for Anet at all three conditions. Conversely, DHM117 exhibited a significant reduction in Anet under eT condition. however, it also demonstrated the ability to recover to ambient levels in the presence of eCO2. This observation becomes a key in understanding the mitigative potential of elevated CO2 on the negative impact of eT on maize, a C4 crop. The interplay between eT and eCO2 emerges as a crucial determinant, resulting in a smaller reduction in net photosynthetic rate as compared to eT alone. This suggests that elevated CO2 has an effective role in counteracting the adverse effects of elevated temperature on maize plants, emphasizing its potential as a protective factor in challenging climatic conditions. In contrast, another study reveals that maize demonstrates signs of CO2 saturation at ambient levels and displays a sluggish response to higher concentrations of CO265. These findings indicate that elevated CO2 can effectively enhance the photosynthetic performance of maize, even under moderately increased temperature conditions. Further, studies revealed the occurrence of photosynthetic acclimation in maize plants following prolonged exposure to elevated levels of CO266. While, in rice a C3 crop, reduced net photosynthetic rate by high temperature was mainly attributed to the reduction of chlorophyll content as well as activities of enzymes involved in photosynthesis67.

Delving further into stomatal conductance (gs) and transpiration rate (Tr), the response of different maize hybrids to elevated CO2 takes centre stage. The presence of elevated CO2 resulted in reduced gs and Tr in 900M GOLD and DHM121, while a slight increase was observed in DHM117 and NK6240. The hybrid DHM121 displayed the lowest variation in gs across the three conditions, indicating that the impact of eT and eCO2 on stomatal response is specific to the genotype. Plants employ diverse responses to adverse environmental conditions, including changes in stomatal function to cope with drought and heat stress68,69. Stomata open for CO2 absorption during photosynthesis but close to prevent water loss through transpiration70. Maize faces the challenge of managing both low water availability and high temperatures in rainfed ecology, leading to a dilemma of preventing water loss while addressing leaf heating. The paradox of water conservation and leaf cooling remains a critical question for maize cultivation under drought and high-temperature conditions.

In maize, the reduction in gs and Tr under elevated CO2 conditions was consistent with previous findings43. The impact of eT on reduction in Tr was found to be significantly more pronounced than its effect on Anet resulting in an increase in WUE in DHM117, while a decrease in other maize hybrids. This divergence in responses among the hybrids underscored the complexity of their reactions to eT and eT + eCO2 conditions. Interestingly, the presence of eCO2, even at higher temperature demonstrated a compensatory effect by recovering Anet while simultaneously reducing Tr. Under eT + eCO2 condition, this dual action contributed to an overall increase in WUE than aT in all four hybrids. The study aligns with previous observations indicating that increased WUE under elevated CO2 can result from an increase in Anet or decrease in gs or a combination of both71. In the specific context of the current investigation, the improved Anet and reduced Tr under eT + eCO2 condition emerged as the primary contributors to the higher WUE. This indicates the intricate interplay of environmental factors and plant physiological responses, emphasizing the need for a comprehensive approach when evaluating the impact of climate change variables on crop performance. The findings underscore the importance of considering multiple variables and their interactions to decipher the complexities associated with the optimization of WUE in maize.

Temperature and humidity play pivotal roles in shaping leaf photosynthetic rates, influencing key processes such as stomatal conductance (gs), and transpiration rate, as well as biochemical processes. Vapor pressure deficit (VPD), affects photosynthetic rates through its influence on leaf stomatal conductance. Stomatal closure mitigates excessive transpiration, preventing a corresponding decline in plant water potential. Importantly, evidence suggests that increasing VPD can inhibit photosynthesis which was also observed in the present study. A decline in transpiration rate (Tr) is typically noted in various crop species under elevated Vapor Pressure Deficit (VPD) conditions72,73,74. The reduction in Tr, resulting from the partial closure of stomata at high VPD, contributes to conservation of soil water. However, this leads to a simultaneous decline in CO2 assimilation due to the synchronization of water vapor and CO2 exchange by leaves and canopies75. Elevated levels of CO2, however, can offset the impact of abiotic stress on water status76. Furthermore, the rise in CO2 diminishes the sensitivity of assimilation rates, caused by high VPD and partial stomatal closure77.

The lipid peroxidation in terms of MDA content has also been used as a valuable stress indicator78,79. In our study, the MDA content of maize hybrids increased significantly under eT as compared to ambient condition. However, the presence of eCO2 mitigated this increase, maintaining MDA levels similar to ambient conditions. There was differential response among hybrids at eT indicating the differential genetic potential among hybrids to cope with climatic stresses. Notably, the hybrid 900M GOLD displayed similar levels of MDA across different treatments, suggesting its resilience and minimal susceptibility to environmental changes. Proline plays a critical role as an antioxidant, osmolyte and the stabilizer of cellular macromolecules and structural components of cell walls80. Elevated temperature induced higher proline accumulation across all hybrids, with 900M GOLD demonstrating comparatively lower proline accumulation, signalling its superior ability to manage cellular activities even under high-temperature conditions. The role of proline in imparting stress tolerance was also reported earlier in rice and maize81,82.

The soluble sugars are also critical in maintaining the cellular structure and growth of plants83. Soluble sugars help in maintaining the leaf water content and osmotic adjustment of plants that is affected by abiotic stresses84,85. Understanding the role of sugars under various abiotic stresses including drought and high temperature is pivotal in modulating several physiological processes86. Previous studies revealed that soluble sugars have a critical role as an osmo-protectant, regulating osmotic adjustment, providing membrane protection, and scavenging toxic reactive oxygen species under various types of stresses87. The starch is also a key molecule in mediating plant responses to abiotic stresses, such as water deficit, high salinity or extreme temperatures. Plants have a general mechanism of remobilizing starch to provide energy and carbon during periods when photosynthesis may be limited, especially under stressed conditions. The dynamics of soluble sugars and starch underpin a pivotal role in maintaining cellular structure and growth. The release of sugars and other derived metabolites serves to support plant growth under stress and acts as osmo-protectants to alleviate the negative effects of the stress88. However, the starch and soluble sugars content were not significantly affected in maize with moderate level of temperature rise and eCO2, but was significantly affected when the temperature was above the threshold level89. In our study, we observed higher accumulation of soluble sugars and starch content under eT and eT + eCO2 in comparison to ambient condition and there was also variation between the hybrids. This suggests that, as water was not a limiting factor, it might have contributed to increased accumulation of soluble sugars and starch, even under eT and eT + eCO2 condition, however it requires further investigation to revalidate these findings. The impact of elevated temperature on biomass and its allocation to vegetative and reproductive components was substantial. Particularly, under the eT condition, there was a significant reduction in stem biomass. However, eCO2 acts as a recuperative force, promoting biomass recovery even under elevated temperature for all four maize hybrids. Our observations align with the hypothesis of positive responses of C4 crops to eCO2, showcasing increased total biomass, further echoing the diverse responses documented in the literature41,42. The impact of elevated temperature (eT) on yield components was also significant, and the extent of this impact varied among different hybrids. Under eT condition, yield parameters such as cob weight, seed number and seed yield was reduced due to poor seed setting. Studies by Johnson90 and Stone91 also revealed that heat stress during pollen formation and pollen shedding is particularly detrimental leading poor seed set and ultimately poor seed yield. The temperature above 30 °C also damages the cell division and amyloplast replication of maize kernels, leading to reduce the grain size and ultimately poor yield92. In the present investigation, we observed differential responses of maize hybrids to eCO2 under eT. The positive impact of eCO2 on C4 crops, specially under eT was significant. The growth and yield components showed positive responses akin to those observed in C3 crops. The presence of eCO2 even under elevated temperature, mitigated the detrimental effects of eT, leading to positive responses in all aspects of phenological stages, physiological processes, biomass production, and overall yield parameters. The present study indicated that 900M GOLD was superior in performance among hybrids under elevated temperature and CO2 which can fetch better return to the farmers under these climatic stresses. Our study aligns with previous research and understanding of these dynamic relationship between plants and their environment, offering valuable cues for sustainable agricultural practices in the face of predicted climatic conditions.

Conclusions

In conclusion, our study demonstrates that elevated temperature significantly affects various traits, spanning phenology, physiology, biochemistry, biomass, and grain yield. The responses of maize hybrids varied with these climatic stresses, where DHM117 and DHM121 exhibiting an increased ASI under eT as compared to NK6240 and 900M GOLD. However, the presence of elevated CO2 reduced ASI similar to ambient condition. The physiological parameters such as MDA and proline increased under elevated temperature but were partially alleviated by eCO2. Among hybrids, 900M GOLD demonstrated superior performance under eT and eT + eCO2 conditions, suggesting potential benefits of cultivating this hybrid for farmers facing climatic stresses. The study contributes valuable insights for sustainable agricultural practices in the context of evolving environmental challenges and breeding programs aimed at developing climate-ready maize hybrids.