Biochemical Response of Maize Plants Grown in the Field Under Different Water Availability: Evaluating the Influence of Leaf Position and Growth Stage

Abstract

Drought is a major abiotic stress factor in agriculture and could greatly affect the production of maize, which is a key food source. Understanding the biochemical response of maize throughout the length of the plant and its life cycle when exposed to water deficit is an important step when exploring new agricultural approaches to minimizing crop losses. In this study, maize plants grown in the field were exposed to three different water regimes (100%, 50%, and 0% irrigation). The biochemical status of the top, middle, and basal leaves was assessed at two different stages of their life cycle (vegetative and reproductive) to evaluate how plants respond to different water deficits. The results showed that, in the presence of water stress, maize development was affected and crop production decreased. Antioxidant enzyme activity, oxidative damage, and osmolyte levels were influenced not only by the irrigation levels but also by the plant section sample. Throughout the maize life cycle, lipid peroxidation, ascorbate peroxidase, and starch levels increased in all leaf sections. However, several biochemical responses are specific to the section: top leaves increase their protein carbonylation, superoxide dismutase, and sugar levels; middle leaves increase their proline and sugar levels; and base leaves increase their superoxide dismutase and proline levels throughout the life cycle. These findings suggest that efforts to minimize the damage caused by water deficits in crop production must consider the different plant sections and phases of the maize life cycle.

1. Introduction

Water is essential for the development and survival of all living organisms, and it is essential to invest in the protection and management of this resource. Climate change predictions of an increase in temperature and decrease in rainfall mean water will become increasingly scarce, increasing drought locations and decreasing irrigation water supplies in many areas of the world [1,2,3]. Water is an essential resource for agriculture, industry, and domestic use, with 70% used in agriculture, 20% in industry, and 10% domestically [4].

Drought, a recurring natural phenomenon, has significant global economic, social, and environmental impacts [5,6] and affects crop yields, food security, and agricultural and forestry losses [7,8]. Agricultural systems are particularly vulnerable to drought, with countries experiencing significant agricultural and economic losses [9,10]. Recent extreme weather events in Europe, such as the hot and dry summers of 2018 [11] and 2022 [12], have highlighted the urgent need for new irrigation systems to adapt to climate change. Several drought indices have been developed to monitor drought conditions and assess their impacts on agriculture and the environment [13,14,15] using meteorological data. Understanding the optimal irrigation strategies for different crops and environments is crucial to reducing agricultural water use and mitigating the effects of water scarcity.

Maize (Zea mays L.) production has increased since 1961, making it one of the most produced crops with the highest production worldwide [16]. Climate change will impact maize crops in several processes (physical, chemical, and biological) owing to their vulnerability to water deficit, decreasing the productivity and nutritional value of the grain [17,18,19,20]. Water stress leads to a reduction in maize yield production during the grain filling (79–81%), vegetative (25–60%), and reproductive (32–92%) phases [21]. Maize plants exposed to water deficit show increased production of reactive oxygen species (ROS)-damaging proteins, lipids, and nucleic acids, which affect growth and yield [21,22]. Abiotic stress targets biological membranes crucial for drought tolerance, and lipid peroxidation is an indicator of membrane instability under stress [23,24,25]. Another indicator of oxidative damage is protein carbonylation, which is an irreversible modification that leads to proteomic changes that affect protein function and structure [26,27]. Higher protein carbonylation levels in plants are associated with aging and stress [26,28].

To balance the oxidative damage caused by biotic and/or abiotic stresses, plants rely on antioxidant systems to protect their cells [29,30]. The increase in the production of antioxidant enzymes, such as superoxide dismutase, catalase, and peroxidase, aids cell detoxification by targeting water deficit-induced ROS production [20,21]. Osmolyte accumulation can also mitigate the impact of stress on maize plants by protecting cellular structures and maintaining osmotic balance, which can be influenced by the water status, growth stage, and crop type [31]. The accumulation of proline and soluble sugars (osmolytes) in maize plants exposed to water deficit helps regulate osmotic pressure, scavenge free radicals, stabilize protein structures, and regulate growth, increasing tolerance to stress [32,33]. Starch is a crucial carbohydrate reserve that balances growth and carbon uptake in plants [34]. Increased starch biosynthesis helps plants cope with water stress, improves photosynthesis, and delays senescence [35].

To better understand and evaluate the impact of two levels of water stress versus normal irrigation in maize plants, it is necessary to use physio-biochemical traits as indicators [20] to assess the ideal percentage of irrigation that preserves the production levels. Moreover, it is also important to clarify whether different parts of the plant are more affected than others and to consider the stage in the life cycle. Therefore, this study aimed to investigate biochemical alterations in plants exposed to three water regimes in the field, while assessing if and how they are influenced by the developmental stage of the plants (vegetative and reproductive phases) and the position of the leaves (top, middle, and base leaves).

2. Materials and Methods

2.1. Growth of Maize with Different Irrigation Levels

Maize (Dekalb DKC6092) was cultivated in three plots (Figure S1), 5 m long, each with two maize lines (distanced 0.75 m), in Estação Experimental António Teixeira, Coruche, Santarém, Portugal (38°56′34.5″ N, 8°30′45.0″ W). Maize was planted along the entire length of the plots using a tractor, and the seeds were 15.6 cm away from one another. The seeds were sown on 14 May 2021 and germinated on 21 May 2021. The soil composition at the study site is presented in Table S1 (data provided by INOVMILHO, Estação Experimental António Teixeira, Coruche, Portugal). To study the influence of drought on maize, one plot received 100% irrigation (600 mm; control), the second plot received 50% irrigation (300 mm), and the third plot received 0% irrigation (0 mm) using a line drip irrigation system. The irrigation system consisted of a watering tube with an internal diameter of 22 mm and drippers placed 20 cm apart, each with a water debt of 1.1 L/h. The field capacity of the study site is 0.33 m³ m⁻³ and the wilting point is 0.11 m³ m⁻³ [36].

Owing to climatic conditions, irrigation was initiated on 26 June 2021 and ended on 18 September 2021. The maize plants were monitored during the growth process. Two samplings were performed: at the vegetative phase, the plants had 12 leaves completely unfolded, stems in the process of elongation, and five internodes formed (13 July 2021), and at the reproductive phase, the plants had a stem completely elongated, nine internodes formed, a flag leaf present, a tassel fully formed, and two ears with husk and silks dried out (24 August 2021). Each sample consisted of three to four leaves from each section of five randomly collected plants from each plot.

2.2. Meteorological Monitorization

Meteorological data from the experimental area (Coruche, Portugal) were obtained from Instituto Português do Mar e da Atmosfera (IPMA). These data were registered by the Automatic Meteorological Station of Coruche (38°56′29.8″ N, 8°30′47.2″ W), located at the experimental site.

2.3. Biochemical Response of Maize

To assess the biochemical response of the maize plants exposed to different water irrigation levels, three to four leaves were collected from the top, middle, and base of the plants sampled in the vegetative and reproductive phases and stored at −20 °C.

2.3.1. Extraction

The samples were cleaned, cut, and homogenized in liquid nitrogen. The homogenous mixture was weighed, approximately 0.2 g for lipid peroxidation, proline, starch, and the remaining parameters (0.5 g). For most parameters, sample extraction was performed with potassium phosphate buffer (50 mM, pH 7.0) at a ratio of 1:6 and with 3% sulfosalicylic acid at a ratio of 2:15 for the other extractions, relative to the sample weight. The samples were macerated in an ice bath, followed by sonication (60 s, 50 amplitude) with a Sonics Vibra-Cell ultrasonic processor (Sonics & Materials, Inc., Newtown, UK) to ensure that the intracellular content was fully extracted. The samples were then centrifuged at 10,000× g for 10 min at 4 °C using a Heraeus Multifuge 3S-R refrigerated centrifuge (Heraeus, Hanau, Germany). The supernatant was collected into a new microtube and stored at −20 °C or used immediately. A Tecan Infinite 200 Pro microplate reader (Tecan Group Ltd., Männedorf, Switzerland) was used for colorimetric assays.

2.3.2. Determination of Soluble Protein Content

To determine soluble protein content, 275 μL of Biuret reagent was added to 25 μL of supernatant in a microplate and incubated for 10 min in the dark at room temperature [36]. Bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) was used as the standard, and the absorbance was read at 540 nm. The results were expressed in mg protein per g of dry weight.

2.3.3. Oxidative Damage

Lipid peroxidation (LPO) and protein carboxylation (PC) were measured to assess oxidative damage in leaves. Based on the protocol described by Buege and Aust [37], LPO levels of the samples were obtained, and lipid peroxidation was determined by measuring the reaction between malondialdehyde (MDA) and thiobarbituric acid (Tokyo Chemical Industry Europe, Zwijndrecht, Belgium), and the absorbance was read at 532 nm. MDA equivalents were calculated using a molar extinction coefficient of 1.56 × 10⁵ M⁻¹ cm⁻¹, and the results were expressed in µmol per gram of dry weight. The protein carbonylation (PC) was determined according to a method adapted from Mesquita et al. [38], by measuring the carbonyl groups (CGs). A molar extinction coefficient of 22 mM⁻¹ cm⁻¹ was used to calculate CGs, and the results were expressed in mmol per gram of dry weight.

2.3.4. Antioxidant Enzymes

To determine the antioxidant enzyme activity, the supernatant was extracted with potassium phosphate buffer. Superoxide dismutase (SOD) activity was determined using the method described by Beauchamp and Fridovich [39]. Samples were read at 560 nm, and one unit was defined as the amount of enzyme required to perform 50% dismutation of the superoxide radical. The results are expressed as U per g of dry weight. Catalase (CAT) activity was determined using the method described by Johansson and Håkan [40]. The absorbance was read at 540 nm, and a standard curve was constructed using formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) standards (5–150 μM). The results are expressed as mU per g of dry weight. Ascorbate peroxidase (APx) activity was determined according to the method described by Nakano and Asada [41]. The samples were read at 290 nm, with continuous reading at 76 s intervals over 20 min. To determine APx activity of APx a molar extinction coefficient of 2.8 m M⁻¹ cm⁻¹ was used, and the results were expressed as U per gram of dry weight. The activity of glutathione peroxidase (GPx) was determined according to the method described by Paglia and Valentine [42]. The absorbance was read at 340 nm, with continuous reading at 76 s intervals over 20 min. To determine the GPx activity of GPx, a molar extinction coefficient of 0.00622 μM⁻¹ cm⁻¹ was used, and the results were expressed as mU per g of dry weight.

2.3.5. Electron Transport System (ETS)

Electron transport system (ETS) activity was determined according to the method described by King and Packard [43]. The absorbance was read at 490 nm, with continuous reading at 25 s intervals over 20 min. ETS activity was determined using a molar extinction coefficient of 15,900 M⁻¹ cm⁻¹, and the results were expressed in mM/min per g of dry weight.

2.3.6. Osmolytes

The proline content was determined using the supernatant from the extraction with 3% sulfosalicylic acid, following the method described by Bates et al. [44]. The absorbance was read at 520 nm, and proline (PanReac Applichem, Barcelona, Spain) standards (1–100 μg mL⁻¹) were used. The results were expressed as U per gram of dry weight. Soluble sugars were determined according to the method described by Dubois et al. [45], using formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) (0.1–3 mg/mL) as the standard. The samples and standard were measured at 492 nm, and the results were expressed in mg/g of dry weight.

2.3.7. Energy Reserves

The starch was quantified in the pellet according to the method described by Dubois et al. [45] after incubation at 95 °C for 1 h in 2.5 mM sulfuric acid (1:2 w/v) (LabChem, Zelienople, PA, USA) [46]. The samples and standard (0.1–3 mg of formaldehyde/mL) were measured at 492 nm, and the results were expressed in mg/g of dry weight.

2.4. Statistical Analysis

Statistical analysis was performed using permutational multivariate analysis of variance (PERMANOVA) with 9999 Monte Carlo tests using PRIMER 6 and PERMANOVA+ [47,48]. Significant differences are considered to be present when p ≤ 0.05 and are identified in figures with different lowercase letters (between maize sections for the same percentage of irrigation), uppercase letters (between different percentages of irrigation for the same maize section), and asterisks (between vegetative and reproductive phases). The global biochemical response of the different leaf sections to different water irrigation levels (100%, 50%, and 0%) in two different phases of the life cycle (vegetative and reproductive) was analyzed by submitting the data (normalized and after resemblance matrix calculation (Euclidean distance)) to an ordering analysis performed by Principal Coordinates (PCOs) and cluster analysis, using the PRIMER 6 and PERMANOVA+.

3. Results

3.1. Plant Status

3.1.1. Growth of Maize Under Different Irrigation Levels

Through the exposure of maize plots to different irrigation levels, we observed a decrease in the height of maize in the vegetative and reproductive phases, with a reduction in the percentage of irrigation (Figure 1A). A significant reduction in maize height relative to that of the control (100% irrigation) was observed in the plot with 0% irrigation during the reproductive phase. The evolution of maize development between samples was similar at different irrigation levels (100%, 50%, and 0% irrigation), which significantly increased growth between 47 and 48 cm (Figure 1A).

The exposure of maize to different irrigation levels did not significantly change the diameter of the maize stem in the vegetative phase (Figure 1B). In the reproductive phase, we noticed that lower water availability (50% and 0% irrigation) reduced the diameter of maize stems (Figure 1B).

3.1.2. Crop Productivity

The irrigation reduction in the maize plots allowed us to observe a reduction in the development of maize cobs, which significantly decreased when the reduction in the maize height relative to the control (100% irrigation) was observed in the plot with 0% irrigation (Figure 2A,B).

3.2. Meteorological Monitorization

3.2.1. Total Precipitation

The data collected by IPMA over the last few years allow us to perceive that, between 1999 and 2003, the total precipitation was on average 487.9 mm per year. A significant decrease in precipitation occurred in 2012 (64.3 mm), and, despite some exceptions (2011, 2014, 2016, 2018, and 2020), between 2004 and 2021, the total precipitation was below 488 mm (Figure S2).

During the experiment (14 May to 30 September 2021), the total precipitation was 76.2 mm (Figure S3). Before the start of irrigation (26 June 2021), the highest precipitation peak (5.572 mm) occurred on 20 June 2021 (Figure S4A). In the time between samplings, the plots were exposed to a total precipitation of 0.49 mm (Figure S3). The second highest peak occurred on 14 September 2021 (26.037 mm), near the harvest (Figure S4A).

3.2.2. Atmospheric Temperature

The data collected by IPMA over the last few years allow us to perceive that, in the Coruche, Santarém district, small temperature variations were observed between 1998 and 2021, with a median maximum temperature of 23.8 °C and a median minimum temperature of 9.0 °C.

During the experiment, the average temperature was 18.9 °C per day throughout the experiment (Figure S3). The average difference between the maximum and minimum temperatures throughout the experiment was 16.9 °C (Figure S4B). Between sampling, the plots were exposed to an average temperature of 23.4 °C (Figure S3). The highest peak occurred on 10 July 2021 (40.9 °C), closer to the first sampling (Figure S4B).

3.2.3. Evapotranspiration

The average evapotranspiration was 901.0 mm (5.30 mm/day) throughout the experiment (Figure S3). The maximum and minimum values of evapotranspiration did not show a significant difference (Figure S4C). Between samplings, the plots were exposed to an average total evapotranspiration of 272.3 (6.33 mm/day) (Figure S3). The highest peak occurred on 14 July 2021 (9.24 mm/day), nearer the first sampling, and the lower peak occurred on 1 June 2021 (2.30 mm/day), after maize germination and before artificial irrigation (Figure S4C).

3.2.4. Modified Palmer Drought Severity Index (MPDSI)

The MPDSI data showed that during maize sowing (May), conditions varied between normal (−0.281) and weak drought (−1.342) (Figure 3). During the maize culture, drought levels increased in August, with the observed values of weak (−1.781) and moderate drought (−2.07). In September, we observed an increase in humidity as the drought changed from moderate to weak (minimum of -1.839 and maximum −1.264). In October, a maximum value of −0.357 MPDSI was observed, but the minimum value was similar to that of the previous month (−1.969) (Figure 3).

3.3. Biochemical Parameters

3.3.1. Soluble Protein

The soluble protein data showed that, in the vegetative phase, the top leaves of maize exposed to the three levels of irrigation had a significantly lower soluble protein than the other sections (Figure 4). In the middle leaves, a decrease in protein when the maize was exposed to 50% irrigation was observed, relative to the other irrigation levels (100% and 0% irrigation). In the base leaves, an increase in soluble protein with a decrease in irrigation levels occurred, a trend also observed for the top leaves.

Regarding the reproductive phase, leaves collected from the three sections did not show a significant difference, except for maize base leaves exposed to 0% irrigation, which showed significantly increased soluble protein in comparison with the other sections exposed to the same irrigation (Figure 4). Interestingly, a reduction in protein levels was observed in the top and middle leaves collected from plants irrigated with 0% irrigation compared to 100% and 50% irrigation. When the maize was exposed to 50% irrigation, a small decrease in the protein content of the base leaves was observed relative to the other irrigation treatments (100% and 0% irrigation).

When we compared the vegetative and reproductive phases, plants without artificial irrigation (0%) had soluble proteins that were significantly different in all sections of maize plants. The plants exposed to other irrigations (100% and 50%) only had the top leaves with soluble protein that was significantly different between the vegetative and reproductive phases (Figure 4).

3.3.2. Oxidative Damage

• Lipid Peroxidation (LPO)

When observing the influence of different irrigation levels on the leaves of different plant sections, we observed that in the vegetative phase, the LPO levels significantly decreased from the base to the top leaves (Figure 5A). The top leaves of maize showed an increase in LPO levels when exposed to 50% irrigation and a decrease when exposed to 0% irrigation. The middle leaves showed an increase in LPO levels with irrigation reduction. The base leaves exposed to 50% and 0% irrigation decreased and increased, respectively, the LPO levels relative to the control (100% irrigation).

Regarding the reproductive phase, leaves collected from the three sections showed significantly increased LPO levels with a decrease in maize height (top, middle, and base leaves) (Figure 5B), similar to what was observed in the vegetative phase. The reduction in irrigation led to an increase in LPO levels in the top and base leaves collected (much higher for the base leaves). In the middle leaves, only the plants exposed to 0% irrigation showed increased LPO levels compared with the control.

When we compared the vegetative and reproductive phases, the plants showed significantly increased LPO levels in the reproductive phase at all irrigation levels (100%, 50%, and 0% irrigation) (Figure 5A,B).

• Protein carbonylation (PC)

In the vegetative phase, a significant decrease in protein carbonylation was observed in the top leaves of maize at all irrigation levels compared with that in the middle and basal leaves (Figure 5C). The top leaves exposed to 50% and 0% irrigation showed increased and decreased PC levels, respectively, relative to the control (100% irrigation), although not significantly. In the middle leaves, a decrease in PC was observed when the maize was exposed to 0% irrigation, when compared to 50% and 100% irrigation, and for the base leaves, a decrease occurred for 50% irrigation, compared to the other irrigation levels.

Regarding the reproductive phase, we observed constant levels of PC in maize plants exposed to 100% irrigation (Figure 5D). When the plants were exposed to 50% irrigation, a decrease in PC was observed from the top to the middle and finally to the base leaves. Maize plants without artificial irrigation (0% irrigation) had decreased PC levels in the middle leaves.

When we compared the vegetative and reproductive phases, the plants without artificial irrigation (0%) had PC significant differences in the top and base leaves of the maize plants. The plants exposed to other irrigations (100% and 50%) had top leaves with PC that were significantly different between the vegetative and reproductive phases (Figure 5C,D).

3.3.3. Antioxidant Enzymes

• Superoxide dismutase (SOD) activity

In the vegetative phase, SOD activity was similar over the entire length of the maize plants, with no significant variation between leaf sections (top, middle, and base) or irrigation levels (100%, 50%, and 0%) (Figure 6A).

During the reproductive phase, we observed a significant increase in SOD activity in the basal leaves of plants exposed to 100% irrigation (Figure 6B). A decrease in SOD activity was observed in the middle leaves in comparison with the other maize sections (top and base) at all irrigation levels, but only in the 50% and 0% irrigation was the decrease significant. In the top and base leaves, we observed an increase in SOD activity with decreasing irrigation.

When we compared the vegetative and reproductive phases, the plants exposed to 50% irrigation showed significantly increased SOD activity in all leaf sections (Figure 6A,B). Maize plants without artificial irrigation (0% irrigation) showed significantly increased SOD activity in the top and base leaves. Under control conditions (100% irrigation), only the basal leaves significantly increased SOD activity in the reproductive phase.

• Catalase (CAT) activity

In the vegetative phase, a decrease in CAT activity in the top and middle leaves of maize was observed with a decrease in irrigation (50% and 0% irrigation) (Figure 6C). The top leaves exposed to 50% and 0% irrigation had significantly lower CAT activity than the other leaf sections (middle and base). The basal leaves had similar CAT activity at all irrigation levels (100%, 50%, and 0%).

During the reproductive phase, we observed similar CAT activity in the same leaf sections of plants exposed to 100% and 50% irrigation (Figure 6D). CAT activity was significantly lower in the base leaves of plants exposed to 100% and 50% irrigation than in the other leaf sections (top and middle). When the maize plants were exposed to 0% irrigation, lower CAT activity was observed in all leaf sections compared to the other irrigation levels (100% and 50%).

When we compared the vegetative and reproductive phases, the plants without artificial irrigation (0%) showed significantly decreased CAT activity in the middle leaves of maize in the reproductive phase. Maize plants exposed to 100% and 50% irrigation had increased CAT activity in the top leaves during the reproductive phase (Figure 6C,D), but only those exposed to the 50% irrigation treatment had significantly increased CAT activity.

• Ascorbate peroxidase (APx) activity

In the vegetative phase, a significant decrease in APx activity with increasing plant height (base, middle, and top leaves) was observed at all irrigation levels (100%, 50%, and 0%) (Figure 6E). In all sections, an increase in APx activity was observed with decreasing irrigation (100%, 50%, and 0%).

Regarding the reproductive phase, a significant increase in APx activity was observed in the top leaves of maize plants compared with the other sections (middle and base leaves) at all levels of irrigation (100%, 50%, and 0%) (Figure 6F). Maize plants exposed to 0% irrigation showed significantly increased APx activity with plant height (base, middle, and top leaves). Plants exposed to 100% and 50% irrigation had similar APx activity when comparing the middle and basal plant leaves.

A significant increase in APx activity was observed in maize plants for all leaf sections and irrigation levels (Figure 6E,F) in the reproductive phase (when compared to the vegetative phase).

• Glutathione Peroxidase (GPx) activity

In the vegetative phase, significantly low GPx activity in the top leaves of maize plants under normal irrigation (100% irrigation) and an increase in GPx activity with a decrease in the level of irrigation (100%, 50%, and 0%) were observed in the top leaves (Figure 6G). GPx activity was significantly higher in the middle leaves of maize in all irrigation treatments, reaching higher values when the maize plants were irrigated with half of the water (50% irrigation). Irrigation levels did not greatly influence GPx activity in the basal leaves.

Regarding the reproductive phase, the GPx activity in the base leaves of maize plants was significantly lower than that in the other sections (top and middle leaves) in the stress levels of irrigation (50% and 0%), and a significant increase in GPx activity was observed in the 100% irrigation (Figure 6H). The top and base leaves of maize plants showed decreased GPx activity with a decrease in irrigation. Plants exposed to 100% and 0% irrigation had similar GPx activities in the middle leaves.

When we compared the vegetative and reproductive phases, significant differences were observed (Figure 6G,H): an increase in activity in the top and base leaves exposed to 100% irrigation and top leaves exposed to 50% irrigation, and a decrease in activity in the middle leaves exposed to 100% and 50% irrigation and base leaves exposed to 0% irrigation.

• Electron transport system (ETS)

In the vegetative phase, significantly low ETS activity was observed in the base leaves of maize plants in all irrigation regimes (100%, 50%, and 0%) relative to the other sections (top and middle) (Figure 7A). The top and middle leaves showed increased ETS activity with increasing water stress, being higher in the lack of irrigation (0% irrigation).

During the reproductive phase, a significant increase in ETS activity along the length of maize plants (from base to top leaves) was observed (Figure 7B). The leaves of maize plants exposed to 50% irrigation had higher ETS activity and lower activity in non-irrigated plants.

When we compared the vegetative and reproductive phases, a significant decrease in ETS activity throughout the length of the plant was observed only during the reproductive phase (Figure 7A,B).

3.3.4. Osmolytes

• Proline

In the vegetative phase, proline had significantly higher values in the top leaves than in the other sections (middle and base leaves) at all irrigation levels (100%, 50%, and 0%) (Figure 8A). When the plants were not artificially irrigated, higher values of proline were observed in all leaf sections (top, middle, and base), relative to the other irrigation levels (100% and 50%).

Regarding the reproductive phase, plants normally irrigated (100%) had significantly decreased proline levels in the middle and basal leaves relative to the top leaves of maize plants (Figure 8B). When exposed to water stress, proline levels were similar along the length of maize plants. With decreased irrigation levels, proline values decreased in the top leaves, were identical in the middle leaves, and increased in the basal leaves.

When we compared the vegetative and reproductive phases, a significant decrease in proline values in the top leaves of maize plants with no artificial irrigation and a significant increase in the middle and base leaves exposed to 100% and 50% irrigation were observed in the reproductive phase (Figure 8A,B).

• Soluble sugars

In the vegetative phase, there was an increase in soluble sugars in all leaf sections, with a decrease in the percentage of irrigation. In the middle and basal leaves, soluble sugar content increased with water stress (Figure 8C). No significant difference was observed between the leaf sections under control conditions (100% irrigation). In the middle leaves, there was a decrease in soluble sugars when plants were exposed to 50% irrigation and an increase in soluble sugars when the plants were exposed to 0% irrigation, compared to the top leaves.

During the reproductive phase, the soluble sugar values were higher in the top and middle leaves (Figure 8D). In the top and base leaves, we observed that soluble sugar content increased with increasing water stress, although the base leaves had significantly lower values of soluble sugar. When the maize plants were exposed to 0% irrigation, a continuous increase in sugar values in the leaves was observed with the height of the plant.

When we compared the vegetative and reproductive phases, a significant increase in sugar content in the top and middle leaves of the maize plants was observed in the reproductive phase at all irrigation levels (Figure 8C,D).

3.3.5. Energy Reserves

• Starch

In the vegetative phase, no significant differences were observed between leaf sections (top, middle, and basal leaves) or irrigation levels (100%, 50%, and 0%) (Figure 9A). However, plants exposed to 0% irrigation had slightly higher starch values in all leaf sections, increasing with the height of the maize plant.

Regarding the reproductive phase, starch values increased with a decrease in water irrigation (100%, 50%, and 0%), and higher values were observed in the top and middle leaves exposed to 0% irrigation (Figure 9B). The base leaves had significantly lower starch content than the other leaf sections (top and middle leaves) when exposed to 100% and 0% irrigation.

When we compared the vegetative and reproductive phases, a significant increase in starch values for all plant lengths was observed in the reproductive phase (Figure 9A,B).

3.3.6. Principal Coordinates (PCOs)

In the vegetative phase, the top leaves were characterized by a decrease in protein, CAT activity, and PC at all irrigation levels. The middle and basal leaves showed lower APx activity and starch values in the vegetative phase at all irrigation levels (Figure 10). A distinction in the biochemical responses of the top leaves from the other sections can be observed in the cluster (Figure S5).

At the highest level of irrigation (100%), an increase in protein and CAT activity in the reproductive phase was observed in all leaf sections. Maize plants exposed to 50% irrigation had increased PC values in the reproductive phase for the top and middle leaf sections. During the reproductive phase, the lack of artificial irrigation was defined by an increase in starch values in all maize leaves (Figure 10). Distinct biochemical responses were observed in the basal leaf during the reproductive phase when exposed to different irrigation levels, causing them to separate in the cluster analysis (Figure S5).

4. Discussion

4.1. Meteorological Monitorization

The profitability and sustainability of agriculture depend critically on favorable meteorological conditions for the development and growth of existing crops [49]. Li et al. [50], observed through a simulation (2008 to 2030) that climate change can have positive or negative effects on maize yields depending on the combination of changes in temperature and precipitation and also that the climatic change impact on maize yields will differ from region to region.

Maize cultivation occurred in months with high evapotranspiration and temperature, and low precipitation, with plants being subjected to severe drought. Constant irrigation is essential, especially during months without precipitation (July and August), to improve crop development and productivity. The importance of a wet climate to the development of maize was detected by Wang et al. [51], who observed positive effects on maize yield when exposed to wet–cold and wet–warm climates and negative effects when exposed to a dry–warm climate. However, the development of maize crops during climate change will not only depend on climatic factors, but also on the evolution and development of technological adaptation, the economic interest of the crops [50], and the availability of water for artificial irrigation.

4.2. Analyses of the Effect of Water Deficit on the Development of Maize Crops

Maize plants are sensitive to water deficits, with a reduction in plant growth and field production in the presence of stress. Water deficits during germination can delay or reduce seedling emergence. Liu et al. [52] observed that the exposure of maize seed to osmotic stress decreased the germination rate and the development of shoots and roots. In our study, the germination of maize seeds was identical for the three conditions performed, because artificial irrigation was only applied after the germination of maize plants, since precipitation was sufficient to maintain the soil moisture for the germination of maize seeds, as indicated by the MPDSI for May.

When water stress sets in during the vegetative phase (May to July), root development allows an increase in plant water uptake, which would help the development of the plant during the reproductive phase (development of tasseling, silking, pollination, and the grain filling stage) when drought is severe and the development of cobs is sensitive to water deficit [53,54]. In our study, we evaluated the difference in the height of maize plants in the vegetative phase and the stem diameter reduction under extreme water deficit (0% irrigation). Ge et al. [55] exposed maize plants in the field to a controlled water irrigation system, and their results showed that stabilization in the growth of the plant at the beginning of the reproductive phase occurs and that the difference in the heights is established in the vegetative phase. A decrease in maize height, leaf area, steam diameter, and the final number of leaves was also observed when the crops were exposed to medium stress (55% field capacity) [55]. Severe water stress (35% field capacity) negatively affects the vegetative phase, leading to a delay in the maturation period of maize plants, and reduces water-use efficiency in the vegetative and reproductive phases [55]. Under water deficit, maize crops are affected, decreasing plant growth and dry weight, number, and quality of the grain, inevitably influencing crop production [56,57,58]. In our study, recovery in the height of plants exposed to water deficit (50% and 0% irrigation) was not observed in the reproductive phase, and the stem diameter was slightly reduced in the presence of water deficit at the reproductive phase.

Although several maize varieties are used in agriculture, exposure to an increasing water deficit will impact their development and production levels [22], especially when stress is felt in the reproductive phase, since nutrient uptake, biomass accumulation, and consequently maize production decrease [55]. For maize production to not be greatly affected by water deficit, irrigation in the flowering and grain filling phases is essential, and artificial irrigation (100%) must be applied [19,59]. In the other phases, the reduction in artificial irrigation in Mediterranean fields does not have a great influence on production [59].

4.3. Biochemical Parameters

The exposure of plants to water deficit activates drought tolerance mechanisms to overcome significant water stress [60]. In this study, we assessed soluble protein, oxidative damage, antioxidant enzymes, and osmolyte accumulation, exploring differences among leaf sections collected in two stages of the maize life cycle under three water availability conditions.

4.3.1. Soluble Proteins

Under limited water availability, during the vegetative phase, maize plants showed increased soluble protein content in all leaf samples, which could indicate a defense mechanism activation by the plant. A previous study observed an increase in total protein content when maize plants were exposed to osmotic stress (−0.49 MPa); however, higher osmotic stress led to a rapid decrease in soluble protein content [61]. Thus, increased protein levels could be an early defense mechanism affected by prolonged exposure (reproductive phase).

A decrease in soluble proteins when plants are exposed to extreme water deficits is observed in the reproductive phase of the top and middle leaves. When exposed to severe water stress, soluble proteins often decrease [20,32,61,62]. The decrease in soluble protein could be due to the rapid degradation of the protein in the presence of water deficit [63]. An increase in drought stress could lead to increased protease activity as a possible response to water deficit, consequently decreasing soluble protein [64]. The increase in protease activity and consequent decrease in soluble proteins contribute to the formation of amino acids essential for osmotic adjustment [65,66]. The increase observed in the base leaves under extreme water deficit could be attributed to drought-induced proteins involved in protecting plant cells.

4.3.2. Oxidative Damage

Increased production of reactive oxygen species (ROS) and membrane damage have been observed in maize plants exposed to water deficit [22,29,62,67]. Abiotic stresses primarily target biological membranes, and membrane stability under water stress is crucial for drought tolerance [23]. Under water deficit, there is an increase in lipid peroxidation of the membranes with a decrease in water availability and from the top leaves to the bottom leaves. These results suggest not only an increase in membrane damage due to the increase in water deficit, but also that the development of the leaves is linked with lipid peroxidation in the cell. Trivedi et al. [58] observed that a decrease in lipid peroxidase could mean a delay in plant senescence and ultimately improve crop production. Plants with lower lipid peroxidation can be considered more tolerant to water deficit [56]; however, tolerant plants also have a competent antioxidant defense system [68]. Under stressful conditions, ROS production increases, causing an increase in lipid peroxidation in plants [56,57,58,69]. Water deficiency increases oxidative damage owing to the generation of free radicals in cells, compromising essential cellular components [24,70]. The exponential increase in lipid peroxidation when plants are exposed to water deficit has been highly correlated with protein oxidation [71,72].

In the presence of ROS, the most common modification of proteins is carbonylation [73], often used as an indicator of oxidative damage at the cellular level [26]. Protein carbonylation is irreversible, causing proteome modifications, changes in protein conformation, and loss of protein activity [27,28]. The increase in protein carbonylation levels in plants has been associated with mechanisms that initiate seed germination, age acceleration, and senescence [26,74]. In the vegetative phase, lower levels of protein carboxylation were observed in the top leaves, probably because they were at an early stage of development. In a review by Ciacka et al. [26], it can be inferred that higher levels of protein carboxylation are associated with older plants, which is supported by our data. Plants exposed to water stress have increased protein carbonylation and consequently accelerate plant aging [26], and an increase in protein carbonylation levels is expected with an increase in plant exposure to stress [28]. In the reproductive phase, there was an increase in protein carbonylation compared to that in the vegetative phase, which is an indication of plant aging. However, in middle leaves exposed to water deficit, a decrease in protein carbonylation levels was detected. A reduction in the level of protein carbonylation may be associated with a decrease in the oxidation of specific proteins, which is not associated with senescence [75].

4.3.3. Antioxidant Enzymes

Exposure of plants to biotic and/or abiotic stresses affects plant development and production because oxidative stress affects plants at the physiological, biochemical, and molecular levels [76]. Plants rely on antioxidant machinery for protection against oxidative stress, and a balance between ROS generation and antioxidant enzyme activity is essential for plant tolerance to stress conditions [29]. To tolerate water deficit, which is responsible for the increased production of ROS, plants have evolved biochemical responses, such as antioxidant and other enzymes responsible for the homeostasis of cellular redox status [77,78].

The exposure of maize plants to water-stressed environments leads to the activation of biochemical responses, increasing SOD, CAT, and APx activity [20,22,62,67,79], and peroxidase activity can sometimes increase [20,62,79] or be maintained [67] in the presence of stress, which could imply slightly different biochemical responses to fight ROS in different maize varieties. Previous studies have reported an increase in antioxidant enzyme activity in maize plants exposed to water deficits [20,56,57,58].

SOD is an important primary line of cellular defense against superoxide radicals and ROS products by their conversion to H₂O₂, which is further detoxified by POD, CAT, and APX [24,80]. In the present study, during the vegetative phase, SOD activity decreased in response to moderate stress (50% irrigation) and increased in response to severe stress (0% irrigation) in the top and base maize leaves. A reduction in SOD activity has been observed in maize leaves under water stress [81]. Lower SOD activity could be a strategy for drought-adapted plants by preventing complete stomatal closure, since reduced superoxide production by the Mehler reaction keeps the stomata slightly open, avoiding complete inhibition of CO₂ fixation [82]. In the present study, during the reproductive phase, SOD activity increased in the presence of both water deficits. Increased SOD activity in plants increases oxidative stress tolerance and has a positive effect on drought stress tolerance [80,83].

Increased activity of H₂O₂ catalyzing enzymes (CAT, APX, and GPx) in maize plants exposed to water stress has been associated with a decrease in ROS, protecting plants from oxidative damage and helping plant development in stressful environments [20,22,29,62,67,79]. CAT is an enzyme with excellent capacity to mitigate oxidative damage [84]. In our study, the exposure of plants to water deficit decreased catalase activity in the top and middle leaves, while maintaining activity in the base leaves. A decrease in CAT activity has been observed in sunflower [85] and maize [29] plants exposed to stress. Jatropha curcas, a drought-tolerant species, reduces catalase activity in the presence of water deficit [86]. A study by Silvia et al. [86] deduced that a decrease in CAT activity would increase peroxisomal H₂O₂, negatively affecting the structure of chloroplasts and compromising CO₂ assimilation. In the present study, we observed drought-reduced CAT activity in maize plants. The increase in APx and GPx activities in all leaf sections during the vegetative phase observed in our study could be a way of maintaining oxidative equilibrium under water deficits [62,86]. The difference in the activity of CAT, APX, and GPx could also be because, although CAT, GPx, and APX all destroy H₂O₂, the Michaelis constant (KM) is different. The KM of CAT is much higher, and thus, it has a low affinity for H₂O₂ [87]. Therefore, CAT will only be active when the other two enzymes do not have the capacity to destroy the H₂O₂ formed. During the reproductive phase, only the base leaves exposed to severe water deficit (0% irrigation) showed decreased APx activity, and a decrease in GPx activity was observed in the top and base leaves in the presence of stress. Considering the above reference, the decrease in APx and GPx activity in the basal leaves of the reproductive phase could be related to the elevated oxidative damage in the membrane cells and the acceleration in the senescence process. The activation of photoprotective mechanisms in plants tolerant to water deficit, such as Jatropha curcas, when exposed to stress can involve an increase in photorespiration, APX, and SOD activities [86]. A study conducted by Egert and Tevini [88] also observed an increase in APx activity in the leaves of Allium schoenoprasum exposed to water stress. Increased GPx activity has been associated with the development and tolerance of plants to abiotic stresses [89]. Maize exposure to herbicides increases GPx activity, which plays an important role in plant detoxification [90]. GPx found in maize cells may be involved in growth regulation, development, and adaptation to various stresses [91]. The general increase in APx and GPx activity in maize leaves during the exposure of maize plants in this study could be related to the activation of antioxidant mechanisms. However, the different levels of enzyme activity in the top, middle, and basal leaves could be related to the stage of leaf development.

4.3.4. Electron Transport System (ETS)

High ETS activity prevents ROS formation through the interaction of molecular oxygen with reduced electron transport components [92]. However, one of the primary sites of ROS production in plant cells is the mitochondrial ETS [92]. In our study, ETS activity increased in maize plants exposed to water deficit in the vegetative phase, demonstrating the extra need for energy for defense in younger plants’ development phase. In the reproductive phase, ETS activity decreased in maize leaves exposed to water deficit. Our results are supported by those of a study conducted by Liu et al. [93], who exposed maize to drought stress and found that ETS was inhibited. It has been observed that water stress changes ETS activity, decreasing electron transfer in sensitive maize varieties, while tolerant varieties suffer small changes in ETS [94].

4.3.5. Osmolytes

Osmoregulation in plants involves increasing the solute content to combat water stress, maintaining turgor pressure and water uptake for growth by lowering the internal water potential. This is achieved through the uptake of inorganic solutes from the soil or the synthesis of compatible osmotic solutes that do not disrupt metabolic reactions [21]. Osmolyte accumulation is essential for osmotic adjustment under water-limited conditions, protecting cellular structures, and maintaining osmotic balance to support water influx. Water status, growth stage, and cultivar influence osmolyte accumulation [31].

Proline, a non-polar amino acid, plays multiple roles in plant stress defense. It helps osmoregulation, scavenges free radicals, and stabilizes protein conformation as a molecular chaperone [21,32]. This protects plant cells from various environmental stressors. Proline accumulation in response to stress is well documented in various plant species, and an accumulation of proline in the leaves increases plant tolerance to stress [95,96]. In this study, plants exposed to severe water deficit showed increased proline levels in leaves in the vegetative phase, with higher values being achieved in the top leaves. Maize plants increase proline levels when exposed to water stress to regulate the osmotic pressure in cells [22]. Previous studies on maize have described increases in proline when inoculated plants were under stress conditions, allowing crop development to limit water availability [32,97]. In the reproductive phase, the increase in proline levels in the middle and basal leaves could not be related to water deficit but to maize plant development [98]. However, the decrease in proline leaves in the top leaves from 0% irrigation may be linked to the response of maize plants to water scarcity. Decreases in proline accumulation under water stress have been associated with stress relief [67], although this could also be a result of plants being unable to synthesize proline.

One of the mechanisms activated by maize plants to tolerate abiotic stress is the increase in soluble sugar content [22,33,67]. Soluble sugars play crucial roles in plants, aiding radical scavenging, osmotic adjustment, carbon storage, and protein structure stabilization. They serve as the main osmolytes for osmotic adjustment, and function as osmoprotectants, growth regulators, and gene expression regulators during abiotic stress [99]. In the present study, maize’s exposure to water deficit led to an increase in soluble sugars in the vegetative and reproductive phases in all leaf sections, with higher levels in the reproductive phase. Bano et al. [32] observed that the exposure of maize plants to water stress increases soluble sugar levels in plant cells. Soluble sugars play intricate roles in cells during normal and stressed conditions, serving as substrates for biosynthesis and energy production, products of metabolic pathways, and regulatory signals [100,101]. They also stabilize membranes and maintain cell turgor, while regulating gene expression [102,103].

4.3.6. Energy Reserves

Starch serves primarily as a carbohydrate reserve in plants and is important for balancing growth and carbon uptake. Starch can be stored for long periods to fuel regrowth or seedling establishment. In photosynthetic cells, a portion of fixed carbon is used to synthesize ’transitory’ starch in chloroplasts, and the functions of starch vary based on the cell type and environmental conditions [34]. Starch is synthesized during the photoperiod and breaks down at night to allow for respiration and growth [104]. Drought increases starch levels, which are then used to release sugars for growth and signaling [34,104], supporting plants during water stress. Previous studies have found that during drought, starch levels decrease in maize plants because it is hydrolyzed to soluble sugar [105,106,107]. In the present study, starch levels increased in maize plants exposed to water deficit in the vegetative and reproductive phases at all leaf positions, with higher levels in the reproductive phase. Our results are supported by a study conducted by AbdElgawad et al. [104], who exposed maize to water stress and observed that starch biosynthesis helps maintain maize leaf growth during drought stress and enhances carbon uptake after recovery. Increases in starch biosynthesis and decreases in protein degradation have been found to enhance photosynthesis and chlorophyll formation and delay maize leaf senescence [35]. In the presence of water stress, the increase in starch synthesis serves as an energy reserve, osmoregulatory support, metabolic process regulation, and protection against oxidative stress [108]. Maize varieties with this ability can tolerate drought better and consequently improve crop development and yield.

Overall, an increase in metabolic mechanism was observed during the reproductive phase, demonstrating a high demand for energy in the development of maize plants. The presence of water stress leads to an increase in lipid peroxidation, indicating oxidative damage, and an increase in APx activity and starch levels, suggesting the activation of defense mechanisms against ROS. Moreover, based on the multivariate analysis (PCO)-based sample distribution, in the vegetative phase, the factor with a higher impact on the biochemical response seems to be the leaf position, whereas in the reproductive phase, it appears to be the level of drought stress.

5. Conclusions

When we examined the biochemical response of maize leaves, the top, middle, and base leaves in most cases presented different values, depending on the leaf position. The biochemical response of these leaves also changes depending on the phase of the life cycle of maize (vegetative vs. reproductive phase). To assess the biochemical response, it is important to consider plant development and leaf position before sampling, with the endpoint protein, CAT, APx, PC, and starch being of particular interest.

This study showed the importance of selecting maize leaves when the objective is to analyze the biochemical mechanisms of maize plants exposed to water stress. Since different biochemical responses occur in different phases, we could infer that, depending on the phase of the life cycle that maize is experiencing water stress, we could require different approaches to mitigating the damage. The importance of water irrigation in situations of lower precipitation and higher temperatures was also highlighted.