Effect of water conditions and nitrogen application on maize growth, carbon accumulation and metabolism of maize plant in subtropical regions

ABSTRACT Drought, water, and nitrogen (N) losses have always been great challenges for agricultural production in subtropical regions of China. To study appropriate irrigation regimes and reasonable N applications, a field experiment was conducted for summer maize (Zea mays L.) from 2018 to 2019. Two irrigation treatments, namely, rain-fed irrigation and supplementary irrigation, were designed, and five levels of N fertilizer were applied. The differences in the growth periods of biomass, leaf area index, agronomic traits, carbon accumulation, and carbon metabolism enzyme activity were measured. Findings revealed that the interaction of water and N has a significant impact on maize growth. Compared to other N treatments, N250 produced significantly higher biomass, leaf area index, agronomic traits, carbon accumulation, and carbon metabolism enzyme activity. In 2018, the agronomic traits and leaf area index were significantly higher than in 2019. Meanwhile, additional irrigation could help improve agronomic traits and the leaf area index. Further correlation analysis revealed that carbon accumulation was positively correlated with carbon metabolism enzyme activity, although lower at maturity than in the flowering period. Overall, the findings suggest that supplementary irrigation in conjunction with N250 treatment is a worthwhile measure for sustainable maize production in subtropical regions of China.


Introduction
Maize (Zea mays L.) has become an important pillar of agricultural development and is the third primary crop in China (Zhou et al. 2021). One of the most important food crops in China is maize, which is grown primarily in subtropical regions. With the development of national green agriculture, maize is playing an increasingly important role in crop production, and the pressure of increasing maize production in the future is high (Yao et al. 2021). Due to drought stress and low N use efficiency (NUE), summer maize production in subtropical regions is facing a huge challenge ). However, due to natural factors, a series of problems, such as intermittent rainfall, high temperatures, and high humidity, will affect the growth of maize (Porter et al. 2014;Welikhe et al. 2016). Water is an important limiting factor for maize production on arable land; at the same time, N is also an important factor for maize growth (Tilman et al. 2002). Drought and N loss are constantly increasing, and whether early or late in the growing season, this can significantly reduce crop yields (Ge et al. 2012;Zhang et al. 2014). Urbanization and the development of high-water-consumption industries and agriculture must be restricted in water-scarce areas (Yuan et al. 2015). Therefore, there is an urgency to strengthen the management of water resources and NUE.
In the subtropical region, it is difficult to increase the irrigation area and the amount of farmland. Long-term dependence on groundwater has caused severe environmental problems Liang et al. 2019). Therefore, proper water storage and use and effective water-saving irrigation have become critical issues (Ali et al. 2019). Different irrigation methods are used in subtropical areas to increase maize yield and efficiently use limited water resources. According to some studies, N fertilizer is the most important input resource for agricultural production, as it improves crop yields and has a positive impact on biomass and maize grain yields (Vos et al. 2005;Javeed and Zamir 2013). Today, the application amount of N fertilizer has far exceeded the suitable range in China (Fan et al. 2013). In many areas of China, the rate of N fertilizer has an impact on groundwater, air pollution, and NUE. (Cui et al. 2018). Therefore, increasing NUE rather than the N input is required for sustained increments in agriculture production, environmental protection, and resource constraints (Hedegaard et al. 2006), and fertilizer input and output balancing have received considerable attention. Increasing the aboveground dry matter yield of summer maize by using multiple inseason N fertilizer applications can improve the utilization rate of N used in research methods (Shaviv 2001;Zhou et al. 2017). According to the findings of Muhammad et al. (2018), who reported that the use of organic N sources (farmyard manure) increased the transportation and application costs.
Previous researchers demonstrated that the application of N fertilizer (Paponov and Engels 2005;Nannen et al. 2011) is closely related to water management (Miao et al. 2011) and has a significant interaction effect (Gheysari et al. 2009;Huang et al. 2014;Jia et al. 2014). These factors affect and extend to carbon and N metabolism, accumulation, transit, and distribution. Therefore, the main purpose of this study is to determine the interaction of different irrigation methods and N fertilizers on spring maize yield, dry matter accumulation, agronomic traits, leaf area index, and carbon and N metabolism and accumulation in different growth periods. The goal is to investigate the impact of different N application gradients on increasing maize yield and improving NUE in subtropical regions under different water irrigation conditions. One of China's most important maize-producing regions is the subtropical region. However, in this region, unsuitable water levels and excessive N supplementation are common, resulting in a decrease in yield and NUE. Therefore, we conducted a fixed-position experiment that examines the effects of different irrigation methods and N fertilizers on maize production under high-yield conditions in the subtropical areas in 2018-2019. Its objective was to determine the interactive effects of different irrigation methods and N on yield, dry matter accumulation, agronomic traits, leaf area index, and the interaction of carbon and N metabolism and its accumulation. The goal is to investigate the effects of different N application gradients on maize yield and NUE under different irrigation methods.

Experimental site description
The field experiment was conducted at the farmland of Guangxi University in Nanning, Guangxi, China (latitude 22°50ʹN, longitude 108°17ʹE, and altitude is 79 meters) from 2018 to 2019. The maize growing season ranged from March to November, with a mean annual precipitation of 1044.6 mm in 2018 and 874.6 mm in 2019 ( Figure 1). According to Chinese Soil Taxonomy, the soil texture in the experimental field from 0-20 cm soil layer was loam, with a pH of 5.4; field capacity of 37.2%; soil bulk density of 1.50 g cm −3 ; soil organic matter of 17.5 gkg −1 ; and available N, phosphorus, and potassium of 126.2, 40.0, and 124.5 mg kg −1 , respectively.

Experimental design
A randomized complete block design with split-plot arrangement was used in the experiment. The water treatments were the main plot, while the N treatments were the split-plot factor. These treatments were replicated three times. The water treatments included supplementary irrigation and rain-fed treatment without irrigation. The five N treatments included control (N0), 150 (N150), 200 (N200), 250 (N250), 300 (N300) kg N ha −1 . Water was supplied to the plots using drip irrigation when the soil volumetric water content in the supplementary irrigation treatment, which was detected by the soil moisture meter of TDR 100 (Spectrum Technologies Inc., Aurora, USA), was less than 28.6%, or equivalently 60% of field capacity. The total irrigation volume during the whole maize growing season, measured by a flow meter, was 86.9 mm and 189.4 mm in 2018 and 2019, respectively ( Figure 1). Sixty-seven percent of total N fertilizers were applied at sowing, with the remaining 33% applied as top dressing at the V12 stage. Furthermore, all plots received 100 kg ha −1 P 2 O 5 and 100 kg ha −1 K 2 O as base fertilizers before sowing.

Maize stem, leaf, and ear biomass
For vegetative growth stages: 6th-leaf stage (V6), 12th-leaf stage (V12), silking stage (R1), milking stage (R3), and maturity (R6). The stem, leaf, and ear samples were separated and oven dried at 105°C for 30 min, and then kept in the oven at 80°C until they reached a constant weight (Zhou et al. 2021). After the constant weight the plant material was kept in the turned off and closed oven for two hours. After cooling, the plant materials were weighed by a sensitive electric balance.

Leaf area index
The leaf area per plant was measured at V6, V12, R0, R3, and R6. Three plants per replicate were selected, and the leaf length and maximum width of each leaf per plant were measured manually using a measuring tape. The leaf area per plant was calculated according to the following formula. Leaf area (cm 2 ) = leaf length × leaf width × Correction factor (0.83)

Agronomic traits of maize
The agronomic traits were counted at R6 using 11 representative samples from an area of 2 m 2 for each plot, and the plant height, stem diameter, and ear height were measured.

Plant carbon accumulation
A plant sample of 0.2 g was taken in a test tube, and 5 mL of potassium dichromate solution with a concentration of 0.1 mol L −1 was added. Subsequently, 5 mL of concentrated sulfuric acid was added to the test tube and gently shaken to mix. The ventilation was opened, the temperature of the digester was controlled at 175°C, and heated for 5 min. A total of 50 mL of distilled water was used to wash the digested sample solution into the Erlenmeyer flask. A total of 5 drops of phenanthroline indicator were added, shaken, and titrated. The carbon accumulation was calculated by following Walkley and Black (1934) using the following formula: Where A is the ferrous sulfate for blank titration (mL), B is the ferrous sulfate used for sample titration, C Fe is the concentration of FeSO 4 standard solution, and m is the sample quality.

Phosphoenolpyruvate carboxylase (PEPCase)
From the flowering period to maturity, three fully expanded leaves or flag leaves were collected in each plot and stored in a refrigerator at −80°C after quick freezing with liquid N. The leaf PEPCase content was measured using an enzyme-linked immune sorbent assay.
Phosphoenolpyruvate carboxylase enzymatic activity is defined as 1 nmol NADH consumed per minute in the reaction system per g of tissue, which is described as a unit of enzyme activity. Frozen plant tissues (−80°C, 0.2 g) were milled and homogenized with 1 mL extract solution and clarified by centrifugation at 8000 × g for 20 min. Absorbance was measured at 340 nm. According to the following formula: where ε is the NADH molar extinction coefficient (6.22 × 10 3 L mol −1 cm −1 ), d is the cuvette light path (0.6 cm), V1 is the total volume of the reaction system (2 × 10 4 /L), V2 is the sample volume in the reaction system (0.02 mL), W is the sample quality (0.2 g), V3 is the extract volume (1 mL), T is the reaction time (5 min), and 10 9 is the unit conversion factor (1 mol = 10 9 nmol).

Sucrose phosphate synthase (SPS)
SPS enzymatic activity is defined as a unit of enzyme activity that catalyzes the production of 1 µg sucrose per minute per g of tissue. Frozen plant tissues (−80°C, 0.2 g) were milled and homogenized with 1 mL of extract solution and clarified by centrifugation at 8000 × g for 10 min. Absorbance was measured at 480 nm. According to the following formula: where C1 is the standard tube concentration (500 µg mL −1 ), V1 is the sample volume to the reaction system (0.01 mL), ΔA1 is the measure of the initial value at 340 nm after mixing well, ΔA2 is the measure of the absorbance again after reacting at 25°C for 30 min, V2 is the extract volume (1 mL), W is the sample's fresh weight (0.2 g), and T is the reaction time (10 min).

Plant sampling and N content determination
The N contents were counted at R6 stage using 3 representative samples from an area of 2 m 2 for each plot, and then oven dried at 80°C in a forced-air oven up to a constant weight and weighted separately. After weighing, the samples were grounded using a cyclone sample mill with a fine mesh (0.5 mm). The following parameters were calculated: Whereas, ANUE and PNUE are the agronomic nitrogen use efficiency and physiological nitrogen use efficiency.

Statistical analysis
All experimental data were analyzed using a two-way ANOVA in SPSS 16.0 (SPSS Inc. Chicago, IL, USA). The least significant difference (LSD) was used to compare the mean value, and it was significant at p < 0.05. The average values of both growth seasons (spring and autumn) were used for the ease of different N and irrigation method compressions in 2018 and 2019. All figures were created using SigmaPlot 10.0 (SPSS Inc., Chicago, IL, USA) and Origin 9.0 (Origin Lab Corporation, USA).

Maize stem, leaf, and ear biomass
The stem and leaf biomass gradually increased with maize growth in 2018 and 2019, and the maximum biomass was observed for N250 at maturity stage ( Figure 2). The leaf biomass was higher than the stem before V6, as opposed to the greater biomass observed in the stem after V6. When the ear occurred at R1, more and more biomass continued to be accumulated in the ear, where the ear increased biomass by 72.55% compared with the stem and by 264.17% relative to the leaf at R6 in two years. Nitrogen-applied treatments played a significant role in enhancing plant biomass (p < 0.05). In comparison with other N levels, N250 and N300 had greater stem, leaf, and grain biomass. Furthermore, in 2019, the biomass apportioned to the ear in N250 is not significantly different from that in N300 (p > 0.05). Water treatment also had a significant effect on biomass after R1 (p < 0.05). Supplementary irrigation contributed more towards biomass, especially the biomass of the ear that was significantly increased by 6.49% in 2018 and 51.39% in 2019 than those in rain-fed treatment (p < 0.05). At R6, the ratio of the biomass of ears to total was 50.90% for rain-fed and 52.06% for supplementary irrigation. The total biomass in the whole plant of maize at R6 was 1729.3 g m −2 in 2018 and 1356.7 g m −2 in 2019.

Leaf area index
In 2018 and 2019, the leaf area index in the growth stage gradually increased, and the maximum leaf area was recorded at R0 (Figure 3). Averaged across the years, the maximum leaf area index, which is 61.8%, 20.69%, 11.69% and 37.23%, higher than V6, V12, R3, and R6, was observed at R0, respectively. N-applied treatments played a significant role in enhancing maize leaf area index (P < 0.05). In the same growth stage, the LAI was increased with N levels. Our results showed that in the rain-fed and irrigation treatments, the LAI in N250 had no significant difference in 2018 (P > 0.05). The rain-fed treatment also had a significant effect on LAI at R0 (P < 0.05). Supplementary irrigation treatment significantly increased the LAI by 5.61% and 17.45% in 2018 and 2019 compared to those in rain-fed treatment, respectively (P < 0.05).

Agronomic traits
Agronomic traits were affected by time, water, N, and their interaction for both years. The interactions were significant for plant height, ear height, and stem diameter (P < 0.05). The existing difference between 2018 and 2019 could be due to climate change. Under rain-fed treatment, N150, N200, N250, and N300 were statistically similar and significantly higher than that of N0. Under supplementary  Figure 5. The effects of rain-fed, supplementary irrigation and nitrogen (N) on PEPCase of maize. Vertical bars represent the standard deviation ± SD (n = 3). Different letters indicate significant differences at P < 0.05. irrigation treatment, plant height, ear height, and stem diameter showed an increasing trend with increased amounts of N fertilizer. Compared with N0, the plant height for N250 was 11.41% and 8.61% higher in rain-fed in 2018 and 2019, respectively, similarly, 12.71 and 10.19% higher in irrigation treatment in 2018 and 2019. Agronomic traits of rain-fed and irrigation treatment were similar and show a gradual upward trend. Plant height, ear height, and stem diameter were not significantly different among N150, N200, and N300 in both years (Table 1).

Carbon accumulation of maize stem, leaf, and ear
Carbon accumulation was distributed in different parts of the maize, where stem, leaf, and ear were the main accumulation parts. Water and N treatment demonstrated a great impact on carbon accumulation during the flowering and maturity stages (Figure 4). Compared with N0, all N treatments significantly increased carbon accumulation in all parts of the maize. Among the N treatments the N250 resulted in higher carbon accumulation. Similarly, the plot receiving supplementary irrigation resulted in higher carbon accumulation compared with rain-fed plots. Averaged across fertilizer, the N250 treatment had significantly higher carbon content in stem, leaf, and grain compared with other N treatments. At the maturity stage in 2018, the carbon accumulation for N150 in maize stem, leaf, and ear was statistically similar to that in N200. Similarly, water treatment also had a significant effect on carbon accumulation. Supplement irrigation contributed much more to carbon accumulation, especially in the ear, which was 11.94% and 35.22% higher than those in rain-fed treatment in 2018 and 2019, respectively (p < 0.05). At the flowering stage, the ratio of carbon accumulation of ear to total was 42.51% for rain-fed and 44.89% for supplementary irrigation, and at maturity, the ratio of carbon accumulation of ear to total was 50.74% for rain-fed and 53.29% for supplementary irrigation.

Phosphoenolpyruvate carboxylase (PEPCase) of maize
Different N and water treatments had significant effects on PEPCase at different growth stages of maize (p < 0.05). During the flowering period, the PEPCase showed the same increasing trend, and the highest value was recorded for rain-fed and irrigation in 2019 ( Figure 5). In N application treatments, N300, N250, N200, N150, and N0 had 24.49%, 30.44%, 19.79%, 18.66%, and 11.84% higher PEPCase in flowering than in the maturity period (p < 0.05). Our results showed that N250 had significantly higher PEPCase for the flowering and maturity periods. Likewise, the flowering period had higher PEPCase than the maturity period in rain-fed and irrigation treatments (p < 0.05). The total of PEPCase content of N0, N150, N200, N250, and N300 were 1. 53, 3.89, 7.16, 22.5, and 14.91 nmol NADH min −1 g −1 of fresh weight under rain-fed treatment, and 2.92, 6.11, 8.83, 19.62 and 12.52 nmol NADH min −1 g −1 of fresh weight in irrigation treatment, respectively. In both treatments, the PEPCase of N250 was significantly higher than those of N0, N150, N200, and N300.

Sucrose phosphate synthase (SPS) of maize
The results showed that both treatments (water and N fertilizer) had a significant effect on SPS ( Figure 6). Among the N fertilizer treatments, N250 had significantly higher SPS than other N treatments for rain-fed and irrigation. The SPS was significantly higher during the flowering period in rain-fed and irrigation treatments than during the maturity period. The SPS trend was N250 > N300 > N200 > N150 > N0 at irrigation, indicating that increasing N application could enhance SPS in the flowering and maturity periods under both rain-fed and irrigation treatments. In both treatments, the N250 had a significantly higher SPS than other N treatments at rain-fed and irrigation (p < 0.05).

Nitrogen use efficiencies
The present experiment revealed that the N use efficiencies in maize were significantly influenced by different irrigation methods and N fertilizer. The agronomic N use efficiency (ANUE) and physiological N use efficiency (PNUE) were remarkably higher in 2018 compared to those in 2019 (Table 2). Our results showed that the ANUE and PNUE were significantly higher in the irrigation plot compared to rain-fed, these results suggesting ANUE and PNUE were 13.8 and 8.15% higher in 2018 compared to 2019, respectively (P < 0.05; Table 2). In addition, the irrigated plot resulted 61.3 and 18.1% higher ANUE and PNUE (P < 0.05) compared to rain-fed plots, respectively. Both ANUE and PNUE showed increasing and then decreasing trend with N application rate, and overall performance were ranked N250 > N300 > N200 > N150, and the differences were significant. These results suggesting that N use efficiency was significantly higher under irrigated plot with combination of N250 treatment than other N treatments.

Discussion
Generally, the final biomass and agronomic traits of maize are highly responsive to different water conditions (Fang et al. 2008;Jia et al. 2014). It has been reported that irrigation at V6, V12, and R0 stages can significantly delay leaf senescence, increase the leaf area index, prolong the milking stage, and increase crop yield (Xu et al. 2018). Our results showed that biomass and agronomic traits in 2018 were significantly higher than those in 2019 (Figures 2 and 4). The development of high-yield potential through increased dry matter accumulation and agronomic features was ensured, perhaps due to excessive rainfall in 2018 (Figure 1), which provided enough moisture to stimulate aboveground biomass in subtropical regions effectively. Under irrigation conditions, higher water use efficiency also played a role in increasing the sink and source of N in maize. Gheysari et al. (2009) showed that the effects of N fertilizer on total aboveground biomass depended on the availability of water in the soil. The stunted plant growth in 2019 had adverse effects on dry matter accumulation than that in 2018 because rainfall is lesser in the former than in the latter. However, the total biomass and dry matter accumulation in irrigation treatments were not statistically different in N250 and N300 treatments, indicating that various N treatments had a greater effect on irrigation and rain-fed crops. These findings suggest that the application of N fertilizer can improve the photosynthetic capacity, N content, carbon content, total biomass, and agronomic traits but decrease the NUE (Peng et al. 2016;Hammad et al. 2017). Zhao et al. (2013) demonstrated that fertilizers could significantly increase biomass and agronomic traits in maize compared with common compound fertilizers. At the same water conditions, it increased with N application levels, and no significant difference was observed between N250 and N300 treatments. Parija and Kumar (2013), reported that the grain yield and plant dry matter are interdependent, further he demonstrated that the plot with high dry matter had a significantly higher grain yield. Leaf area index is a significant determinant of plant development, dry matter accumulation, and carbon fixation. The increase in LAI is possibly due to the growth of new leaves or the enlargement of expansion leaves (Mandal et al. 2005). Our results suggest that higher LAI with increased N application could be attributed to significant increases in leaf expansion (length and breadth) that resulted from cell division and cell enlargement at higher N rates (Shafi et al. 2011;Kar and Kumar 2015). Leaf senescence in the late stage of crop growth is an important reason for the decrease in LAI (Bandyopadhyay et al. 2010;Thomas 2013;Pradhan et al. 2013). The important sources of carbon in maize are stem and grain, whereas the interactive effects of different water regimes and N applications on stem, leaf, and grain in summer maize showed a significant increase (Figure 4). The reason for this increase is that optimum water-nitrogen interactions might effectively improve the photosynthetic capacity of ear leaves in summer maize, as well as plant production capacity and photosynthetic allocations (Srivastava et al. 2018). During heavy rainfall in 2018, carbon accumulation in each part of the maize increased with nitrogen input. Carbon accumulation was higher in the irrigation treatment than in the rain-fed treatment, especially in the maturity stage. Carbon accumulation could be at its peak in 2019 in irrigation treatment (Figure 4). Carbon accumulation was significantly higher in irrigation treatments with N250 nitrogen than in other N rates, indicating that this interaction has a favourable carbon accumulation environment. Previous studies reported that the management of water and nitrogen is closely related to the accumulation and distribution of carbon, and maize carbon metabolism enzyme activity plays a role in the entire growth process (Zhou et al. 2020). Researchers demonstrated that SPS is a multifunctional protein and a key enzyme in plant metabolism and performs various biological functions during plant growth. Sinclair and Rufty (2012) reported that moderate water conditions were required from the flowering to maturity stage to ensure the absorption and utilization of N in the soil. This condition improved plant growth and nitrogen usage by increasing carbon accumulation in the stems, leaves, and grains, as well as PEPc and SPS activities. Under irrigation and rain-fed conditions, the carbon metabolism enzyme activity increased with the N application, and at N250 treatment, reached maximum ( Figures 5 and 6). The interactive effects of water and N are beneficial to carbon accumulation and carbon metabolism enzyme activities, which can provide more nutrients during the maize growth period. The carbon accumulation of each treatment in different parts exhibited clear differences because of the effects of the different waternitrogen interactions. Low rainfall in 2019 limited N release capacity under low-moisture conditions in the flowering stage, probably due to a lack of mineral N availability and inhibited root activity in the soil (Shao et al. 2013). Nitrogen participates in morphological changes during the vegetative period, thereby affecting carbon accumulation. Despite the lack of water, carbon accumulation gradually increases in irrigation conditions over the maturity stage, showing that the N in the soil has been fully absorbed (Teixeira et al. 2014).
Carbon accumulation and carbon metabolism enzyme activity increase with N application under the same water conditions; likewise, water content increases at the same N level (Guo et al. 2016). The activities of carbon accumulation and metabolic enzymes are much higher during the flowering stage than in the maturity stage, possibly due to plant senescence, which may reduce the content in the maturity stage.
Due to relatively high rainfall in 2018, nitrogen release capacity gradually increased with increasing N fertilizer rates and played a remarkable role in the absorption and utilization of N. The results of the present study showed that the ANUE and PNUE were significantly higher during 2018 as compared to 2019, these increased might be due to high rainfall in 2018. Moreover, the ANUE and PNUE were statistically higher in the irrigated plot than in the rain-fed plot (Table 2). Previous researcher reported that the water and N interaction could promote N absorption and utilization (Gheysari et al. 2009;Jia et al. 2014;Guo et al. 2016). Likewise, summer maize's growth and development may require a coordinated relationship between water and N in order to improve N accumulation in various organs and then promote accumulation in the ears (Li et al. 2017). The current results showed that the ANUE and PNUE were significantly higher in the N250 treatment under irrigated plots compared to other N treatments.
Furthermore, interactions between water and N fertilizer could increase biomass, agronomic traits, leaf area index, and plant growth. When spring maize was irrigated during the flowering season and treated with N250, it resulted in higher carbon accumulation and metabolic enzyme activity in the maize plant. However, N migration mechanisms in the soil under different water conditions influenced the maize plant growth. Further study is needed to investigate these mechanisms in different climates and fertilizers.

Conclusions
Nitrogen and different irrigation treatments had significant interactive impacts on biomass, agronomic traits, leaf area index, carbon accumulation, and carbon metabolism enzyme activity in summer maize. Managing the optimal interactions of N and different irrigation treatments was beneficial to maize growth; regardless of irrigation treatments, the enhancement of summer maize under N250 and N300 treatments was much higher. Although the rain-fed treatment can boost summer maize growth and boost metabolites, it does not have the same impact as the supplementary irrigation treatment. This study found that the N fertilizer application rate of 250 kg N ha −1 was the best treatment for rain-fed and supplemental irrigation treatments. We suggest that an appropriate N fertilizer is more feasible under supplemental irrigation in subtropical areas of China. At the same time, compared with N300, N250 can reduce fertilizer costs and the risk of groundwater pollution.