Inter-subspecies diversity of maize to drought stress with physio-biochemical, enzymatic and molecular responses

Conducting the experiment

The soil utilized in this study was obtained from the experimental research area of Eskişehir Osmangazi University at a depth of 0–30 cm. It was prepared by sieving through a 2 mm filter after allowing it to dry in the open for a week. The soil is free of salt and has a sandy loam texture. The pH of the soil is 7.99, indicating an alkaline nature. Its organic matter content is low, at 1.08%. Additionally, the soil is calcareous, with a calcium content of 1.98%. It contains adequate levels of potassium, copper, and iron, but not enough phosphorus, manganese, or zinc.

One kilogram of soil was saturated with water and placed in a container with perforations at the base to ascertain the field capacity of the soil. After a 24-h period, the water that gravitationally drained was collected, and the field capacity of the soil was determined using the difference method, as outlined in the following formula (Danish et al., 2020).

$$Fieldcapacity\left(\mathrm{\%}\right)={\displaystyle \frac{Wateradded\left(ml\right)-Waterinfiltratedin24hours\left(ml\right)}{Soilweight\left(g\right)}\times 100}$$

The study employed three distinct subspecies of maize: dent maize (Zea mays indendata, cv. P0937), popcorn maize (Zea mays everta, cv. Baharcin), and sugar maize (Zea mays saccharata Sturt, cv. Adapare). The maize varieties utilised in the study are commonly cultivated in Türkiye and are known as drought tolerant.

In order to investigate the drought tolerance of the intersubspecies, the ones of every subspecies that were drought-tolerant were chosen.

Ten-liter plastic containers were utilized to cultivate maize seedlings, containing a mixture of 2:1:1:1 soil, sand, peat, and perlite. A basic solution containing macro- and micro-elements was used for fertilization of the pots. The solution included 200 mg of nitrogen (NH₄)₂SO₄, 125 mg of potassium, 100 mg of phosphorus (KH₂PO₄) per kg of soil, 2.5 mg of iron (Fe-EDTA), and 5 mg of zinc (ZnSO₄.7H₂O) per kg of soil. A total of 12 maize seeds were planted in each pot for each treatment and replication. Following the emergence of the plants, the number of plants per pot was reduced to six. The plants were maintained at a temperature of 25/17 °C, with a light intensity of 800 µmol m⁻² s⁻¹, a humidity of 65%, and a light/dark cycle of 14/10 h throughout the duration of the experiment. The Hoagland solution was applied once a week until harvest, comprising 5 mM KNO₃, 1 mM KH₂PO₄, 5 mM Ca(NO₃)₂, 2 mM MgSO₄, 50 μM H₃BO₃, 10 μM MnCl₂, 1 μM ZnSO₄, 0.4 μM CuSO₄, 0.1 μM H₂MoO₄, and 20 μM Fe-EDTA.

The maize plants were grown under standard irrigation conditions, which included maintaining 80% field capacity for the initial 14 days following germination and emergence until the V3 stage. Throughout the drought trial, the moderate drought group was maintained at 50% field capacity, the severe drought group at 30% field capacity, and the control group was maintained at 80% field capacity. From the time of plant emergence until the V14 stage, which denotes the end of the vegetative cycle, the plants were subjected to 50 days of drought treatments. Namely, the maize plants were exposed to drought stress from V3 to V14. At the conclusion of the designated period, the plants were harvested for subsequent analysis.

Osmoregulation parameters

In order to determine the RWC and LOT values of the plants, three discs with a diameter of 2 cm were removed from the leaf samples. The fresh weights (FW) immediately, the turgor weights (TW) after being kept in pure water for 4 h, and the dry weights (DW) after being kept at 70 °C for 24 h were recorded. The data obtained were used to calculate the RWC and LOT values using the following formulas, with the resulting values expressed as a percentage (Gulen & Eris, 2003).

$$\mathrm{R}\mathrm{W}\mathrm{C}=(\mathrm{F}\mathrm{W}-\mathrm{D}\mathrm{W})/(\mathrm{T}\mathrm{W}-\mathrm{D}\mathrm{W})\times 100$$

$$\mathrm{L}\mathrm{O}\mathrm{T}=(\mathrm{T}\mathrm{W}-\mathrm{F}\mathrm{W})/\mathrm{T}\mathrm{W}\times 100$$

Proline analysis was conducted in accordance with the methodology described by Bates, Waldren & Teare (1973). For the analysis, 0.2 g of plant leaves were homogenized with 4 mL of 3% sulfosalicylic acid and centrifuged (Neofuge-13R) at 6,000 rpm for 10 min. Two milliliters of the supernatant were transferred to glass test tubes and 2 mL of glacial acetic acid and 2 mL of acid ninhydrin were added. The tubes were covered with aluminum foil and placed in a water bath at 100 °C for 1 h. The samples were then cooled in an ice bath for 10 min. Four milliliters of toluene were added to the cooled samples, which were then shaken gently and left for 5 min. Once two phases were observed in the tubes, samples were taken from the upper phase and concentrations were determined at 520 nm with a spectrophotometer (Thermo-Aquamate) on the calibration curve prepared previously using PRO standards. The amount of PRO was calculated using the formula: [(µg proline/ml × ml toluene)/115.5 µg/µmol]/[(g sample)/5] = µmol proline/g fresh weight.

The soluble protein (SPR) concentrations of leaf samples from all treatments were determined using a bovine serum albumin (BSA) standard according to the method of Bradford (1976). The absorbance values of the solution were read at a wavelength of 595 nm in a spectrophotometer (Thermo-Aquamate). The amount of SPR was calculated against a standard curve generated using BSA standards.

Membrane injury parameters

The MDI in the leaves of the plants was determined according to the methodology described by Sairam, Shukla & Saxena (1997). For this purpose, the leaves were harvested at random from the plants in each condition and carefully cut with a scalpel. The leaf samples were then washed thoroughly with pure water at least six to seven times. This procedure ensured that cellular fluid contamination from the cut portions of the leaves did not affect the MDI values. Subsequently, 2 cm leaf sections were carefully excised from the aforementioned samples, placed in glass tubes, and 20 ml of pure water was added. The samples were then shaken on a shaker for 4 h, after which the relative ion amount (A) passing into pure water was measured with an electrical conductivimeter (S230-K; Mettler-Toledo). Then, the identical samples were maintained in a 100 °C water bath (Nuve, Turkey) for 30 min, then allowed to cool to room temperature before being subjected to a second measurement of the relative ion content (B). Finally, the MDI of the leaves was calculated using the following formula:

$$\mathrm{M}\mathrm{D}\mathrm{I}=(1-\mathrm{A}/\mathrm{B})\times 100$$

The extent of lipid peroxidation in the leaf tissues of the plants in the control and stress groups will be quantified by measuring the amount of MDA using the method described by Hodges et al. (1999). The method entails the homogenization of 0.1 g of fresh leaf samples taken from the leaves of the control and stress groups in three replicates with 1.5 ml of 5% trichloroacetic acid (TCA) on ice after being cut into small pieces. The aforementioned mixture was then centrifuged at 12,000 rpm at 25 °C for 15 min, after which the supernatant was utilized to ascertain the quantity of MDA. Equal volumes of the supernatant and a 20% TCA solution containing 0.5% thiobarbituric acid (TBA) were transferred to new tubes. Subsequently, the tubes were placed in a water bath at 95 °C for 30 min, after which they were transferred to an ice bath to halt the reactions within the tubes. Subsequently, the tubes were centrifuged at 1,000 rpm for 5 min, and the absorbance was measured at 532 and 600 nm wavelengths in a spectrophotometer. A 20% TCA solution containing 0.5% TBA will be employed as a blind. The MDA content (nmol gFW⁻¹) in leaf tissues will be calculated according to the following formula:

$$\mathrm{M}\mathrm{D}\mathrm{A}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}=[(\mathrm{A}532-\mathrm{A}600)\times \mathrm{e}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}]/[155\times \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{y}]$$

The H₂O₂ levels were determined in accordance with the methodology described by Velikova, Yordanov & Edreva (2000). For this purpose, leaf tissues (500 mg) were homogenized with 5 mL of 0.1% TCA. The homogenate was then centrifuged at 12,000 g for 15 min. A volume of 0.5 mL of the supernatant was combined with 0.5 mL of a 10 mM potassium phosphate buffer solution (pH 7.0) and 1 mL of 1 M KI. The absorbance of the supernatant was measured using a spectrophotometer with a wavelength of 390 nm. The quantity of H₂O₂ was determined by means of a standard curve.

Antioxidant enzyme activities

The antioxidant enzyme activities (SOD, APX, and CAT) were analyzed according to the method described by Cakmak & Marschner (1992).

For the extraction of the enzymes, 0.2 g of plant leaves were homogenized in 2 mL of K-P buffer (pH 7.6) and centrifuged at 15,000 g for 20 min at 4 °C. Subsequently, the supernatant was removed, and the reactions were prepared for each enzyme activity, as detailed below. The supernatant obtained from the aforementioned extraction was utilized for all enzyme activity readings. As enzyme activities were calculated in relation to protein content, it was first necessary to determine the protein content.

To determine SOD activity, the following solution was prepared: 2.9 mL K-P buffer (pH 7.6), 0.5 mL sodium bicarbonate, 0.5 mL methionine, 0.5 mL nitro blue tetrazolium chloride (NBT), 0.5 mL riboflavin, and 0.1 mL sample supernatant. This solution was then placed in glass test tubes. The tubes were gently mixed and then incubated under light for 10–15 min (until the color turned blue). The absorbance at 560 nm was then determined with a spectrophotometer. The quantity of enzyme was calculated in micromoles per milligram of protein.

For the determination of APX activity, 0.7 mL of K-P buffer (pH 7.6), 0.1 mL of H₂O₂, 0.1 mL of the sample supernatant, and 0.1 mL of ascorbic acid were added, and absorbance readings were taken at 290 nm for 1 min in a spectrophotometer. The enzyme amount was calculated as micromoles per milligram of protein per minute.

The CAT activity was determined by adding 0.8 mL K-P buffer (pH 7.6), 0.1 mL sample supernatant, and 0.1 mL H₂O₂, and absorbance readings were performed at 240 nm for 1 min. The amount of enzyme that decreased the absorbance by µmol in 1 min at 25 °C was calculated as nmol/mg protein/min.

HSP70 and HSP90 gene expression levels

The RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) was used to isolate total RNA from 100 mg of ground-up leaves that had been frozen in liquid nitrogen. Following the isolation of RNA, the purity of the RNA was quantified using a nanodrop device (Thermo ND2000, Waltham, MA, USA). RNA samples with an A260/A280 ratio of approximately 2.0 were utilized for cDNA synthesis. Total RNA was isolated once more from samples exhibiting values below the desired threshold, and this process was repeated until the requisite value was achieved. The measured RNA concentrations were adjusted to 1,000 ng/µL, and cDNA was obtained by reverse transcription polymerase chain reaction (RT-PCR). The application of RQ1 RNase-free DNase (Promega, Madison, WI, USA) eliminated any DNA contamination. For cDNA synthesis, Procomcure Biotech, Thalgau, Austria, provided the VitaScript TM First-strand cDNA Synthesis Kit (PCCSKU1301). The cDNA samples were then adjusted to 100 ng/µL and the expression levels of the gene products of interest were determined by RT-qPCR (real-time PCR). RT-qPCR conditions: 95 °C 5 min, followed by 40 cycles of 95 °C 15 s, 60 °C 30 s, and 72 °C 30 s. Following the synthesis of cDNA from the prepared RNA samples, a RT-qPCR analysis was conducted using specific primers for the selected genes and maize Actin as a housekeeping gene (Table 1). The study was conducted using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the 2X Magic SYBR Kit (Procomcure, Thalgau, Austria). The 2^−ΔΔCt technique was employed to calculate the relative expression (Livak & Schmittgen, 2001). Three biological and two technical replicates were employed for each analysis.

Western blotting for HSP70 and HSP90

The purification of plant samples was conducted in accordance with the methodology proposed by Abraham-Juárez (2019). The Bradford technique was utilized to quantify the protein concentration of the samples. Based on the identified protein concentrations, the sample amounts were adjusted. Subsequently, the samples were subjected to electrophoresis on a 12% polyacrylamide gel. Subsequently, a PVDF membrane with an area equivalent to that of the gel was submerged in 10 mL of 100% methanol for 1 min in a square Petri plate. The transfer procedure was conducted using the ThermoFisher Scientific iBlot 2 Dry Blotting System. The gel-to-membrane transfer procedure was conducted using the sandwich-shaped membrane transfer mechanism of the iBlot 2 device at 20 volts for 7 min. A blocking solution was subsequently formulated following the completion of the transfer procedure. Later, the gel image was detached from the transfer paper and immersed in the pre-prepared blocking solution for a period of 5 min. The blocking solution was then transferred to 2.5 ml tubes, with 2 ml in each tube, which was equivalent to the quantity of antibodies employed. This was for the antibody application procedure. In this investigation, Agrisera UBQ11 (internal control), HSP70, HSP90, and HRP Goat Anti-Rabbit IgG (H + L) were used as secondary antibodies. Then, the iBind flex card was attached to the membrane cylinder, after which 10 milliliters of blocking solution were wetted. The membrane was immersed in the ECL solution for a period of 2.5 h. A chemiluminescence imaging device was utilized in the imaging procedure. Invitrogen SeeBlue Plus2 Prestained Standard was used as a ladder. Prior to imaging, the membrane was incubated in a 1:1 ECL solution for 5 min in the dark. The final band images from the films were converted to digital media by means of a scanner. A densitometric analysis of the bands was conducted using the American Institute of Health’s “Image J” tool (http://rsb.info.NIH.gov/nih-image/).

Statistical analyses

The study data was subjected to an entirely random factorial design in order to examine for variance using IBM SPSS 26. To identify differences in means, the Duncan multiple comparison test was utilized. The data is presented in bar graphs and error bars, which display the mean and standard error of the data. The figures were generated using IBM SPSS 26. Correlations between the examined parameters were analyzed by Pearson correlation analysis according to p < 0.05.

Osmoregulation parameters

A statistical analysis revealed that maize subspecies, drought treatments, and their interactions were found to be statistically significant at the 1% level in terms of leaf RWC, LOT, PRO, and SPR content (Table S1).

In the absence of stressors, the RWC of maize subspecies exhibited the highest levels in sugar maize, reaching 95.20% (Fig. 1A). The water content of popcorn and dent maize was 77.48% and 72.43%, respectively. In the context of the moderate drought, the water content of dent maize showed the highest level of resilience, despite exposing the lowest water content under normal conditions. The reduction in water content of dent maize was approximately 4%, while popcorn maize displayed a 35% decline and sugar maize displayed a 44% reduction. In response to severe drought conditions, sugar maize had a 70% reduction in water content compared to the control, while popcorn maize demonstrated a 35% decline in water content at the same rate as moderate drought severity. However, dent maize lost the content of water by approximately 15% compared to the control, resulting in a RWC value of 61.82%. The LOT in maize plants under drought stress was found to be parallel to the RWC. In the dent maize, a 33.42% LOT was observed under severe drought stress. However, even under moderate drought stress, turgor loss exceeding 40% was measured in popcorn and sugar maize. Although turgor loss in sugar maize under severe drought remained at the same level as in moderate drought, LOT in sugar maize reached 62.93% (Fig. 1B).

As drought stress increased, PRO accumulation increased in dent and popcorn maize, however decreased in sugar maize (Fig. 1C). Dent maize, which had the lowest PRO content (12.13 mmol g⁻¹FW) under control conditions, reached a PRO value of 35.99 mmol g⁻¹ FW when exposed to severe drought. Popcorn had a higher PRO content than other maize subspecies under both control and moderate drought conditions. In contrast, PRO content reduced in sugar maize as a response to drought stress, reaching a minimum of 1.56 mmol g⁻¹ FW in severe drought. The SPR content of maize leaves decreased under drought stress across all maize subspecies. The highest SPR content was observed in sugar maize under all conditions; however, it was the lowest in dent maize. The greatest reduction of SPR in maize leaves belonged to dent maize under moderate drought stress and in sugar maize under severe drought stress in comparison to normal conditions. The highest SPR content was measured in popcorn maize under severe drought stress, in comparison to moderate drought stress (Fig. 1D).

Membrane injury parameters

The results of variance analyses of membrane injury parameters (Table S1) indicated that there were statistically significant changes in membrane damage (MD%), MDI, MDA, and H₂O₂ content among maize subspecies, as well as in response to drought stress. The interaction between maize subspecies and drought stress was found to be insignificant for MD% and MDI. However, significant interactions were observed for MDA and H₂O₂ content.

The greatest degree (19.36%) of MD%, as indicated by electrolyte leakage, was observed in sugar maize subjected to severe drought stress. Membrane damage increased with the severity of drought stress in all maize subspecies. Dent maize indicated the least damage (1.20%) under moderate drought stress, while popcorn maize exhibited the least damage (3.60%) under severe drought stress (Fig. 2A). As anticipated, the MDI demonstrated a decline in accordance with the severity of the applied stress. The MDI calculated a range of values, spanning from 65.44% (sugar maize under severe drought stress) to 81.16% (sugar maize under control conditions). The MDI values were close in popcorn maize in all conditions. Notwithstanding the fact that the highest MDI was observed in sugar maize under control conditions, it was found in popcorn maize under moderate and severe drought (Fig. 2B).

The MDA values of maize plants under control conditions ranged from 7.89 to 9.21 nmol gFW⁻¹ (Fig. 2C). Popcorn maize had the lowest MDA values, while dent maize had the highest. When maize plants were exposed to moderate drought stress, the MDA values increased by 22.34% in dent maize, 85.30% in popcorn maize, and 29.73% in sugar maize. As the severity of the drought increased, the MDA content increased by 7.42%, 12.95% and 24.63% to 12.03 nmol gFW⁻¹ in sugar maize, 12.72 nmol gFW⁻¹ in dent maize, and 18.22 nmol gFW⁻¹ in popcorn maize, respectively. The highest increase was in popcorn maize at both stress severities. Since MDA content was high in dent maize under normal conditions, the increase in stress conditions was lower than in the other subspecies. Nevertheless, the MDA content of sugar maize was comparatively lower increase when the drought severity was altered from moderate to severe.

The lowest levels of H₂O₂ were observed in dent maize, while the highest levels were in popcorn maize under all conditions (Fig. 2D). Even though the levels were low, the increase in H₂O₂ content was more pronounced in dent maize as drought stress intensified compared to other subspecies. In the absence of drought stress, the H₂O₂ content of dent maize was 11.14 nmolg⁻¹FW, that of popcorn maize was 37.59 nmolg⁻¹FW, and that of sugar maize was 32.44 nmolg⁻¹FW. When moderate drought stress was imposed, the H₂O₂ content of dent maize increased by 10.65 units. This increase is 4.30 units in sugar maize. In the case of severe drought conditions, the H₂O₂ content increased by 13 units in popcorn maize in comparison to normal conditions. In contrast, it increased by only 3.49 units when the drought changed from moderate to severe, representing the lowest increase among the subspecies.

Antioxidant enzyme activities

A significant change was observed in the antioxidant enzyme activities of maize plants exposed to drought stress. The interaction between maize subspecies and drought stress was not significant for SOD activity (Table S1). The results of the study indicated that all antioxidant enzymes examined exhibited a parallel increase in the severity of drought stress in all maize subspecies (Fig. 3). The greatest increase in SOD enzyme activities was observed in dent maize plants under moderate drought stress. In severe drought conditions, the highest SOD activity was observed in popcorn and sugar maize. Dent maize revealed similar SOD activity under both moderate and severe drought conditions.

The increases in APX activities of maize plants were close between dent and popcorn maize under moderate drought conditions in comparison to control conditions. However, the increase was twice as great in sugar maize. Severe drought resulted in a 2.6-fold increase in APX activity in sugar maize in comparison to control conditions, while it increased 1.8-fold in popcorn maize and 1.4-fold in dent maize (Fig. 3). The activity of CAT increased rapidly in dent and sugar maize under moderate drought conditions, while it increased more slowly in popcorn maize. In the case of severe drought, the highest CAT activity was observed in sugar maize. In severe drought, CAT activity increased by more than fourfold in sugar and popcorn maize compared to the control. In the transition from moderate to severe drought stress, CAT activity increased by a factor of 2.7 in popcorn maize. Although the magnitude of the increase was considerable, CAT activity was the lowest in popcorn maize (Fig. 3).

HSP70 and HSP90 gene expression levels

The expression of the HSP70 gene differed among maize subspecies, however was not significant for drought stress or the maize subspecies × drought stress interaction. In contrast, the expression of the HSP90 gene was statistically significant for all experimental variants (Table S1).

The expression levels of the HSP70 gene indicated notable differences among maize subspecies under conditions of drought stress (Fig. 4). In dent maize, the expression level increased from 1.00 to 1.52 under moderate drought stress and decreased to 1.31 under severe drought. In popcorn maize, there was a gradual increase in gene expression levels with increasing drought severity, with levels being slightly higher than those observed in other maize subspecies under all conditions. In contrast, the level of HSP70 gene expression in sugar maize showed a decrease with increasing drought severity, reaching a highly low level.

The level of gene expression of HSP90 was significantly elevated and overexpressed in sugar maize under conditions of severe drought. In dent maize, the gene expression level decreased from approximately 1.00 under normal conditions and moderate drought to 0.71 under severe drought. In popcorn maize, the expression of HSP90 increased by a factor of 1.5 in the presence of moderate drought in comparison to normal conditions and decreased by a factor of 0.8 in the presence of severe drought. In sugar maize, expression levels demonstrated a marked increase with the progression of drought severity. The HSP90 gene in sugar maize expressed a 3.4-fold increase under moderate drought conditions and a 17.7-fold increase under severe drought conditions relative to normal conditions.

Western blotting for HSP70 and HSP90

Western blot analysis revealed that the band intensity of the HSP70 protein was highest in dent maize under normal conditions (Fig. 5). The next highest level of HSP70 protein was observed in sugar maize, although this was considerably lower in popcorn. Under the moderate drought, the band intensity of HSP70 was found to be relatively similar in dent and popcorn maize, while exhibiting a higher level in sugar maize. In severe drought, the band intensity of HSP70 was ranked from largest to smallest in dent, sugar, and popcorn maize. The manifestation of changes in HSP70 protein expression under drought stress differed between maize subspecies. HSP70 expression indicated a gradual increase with drought in popcorn maize, however a decline in sugar maize.

The intensity of the HSP90 protein band, which is typically high in dent maize, was found to be equivalent in popcorn and sugar maize and approximately half that observed in dent maize. Moderate drought stress resulted in a reduction in the quantity of HSP90 protein in dent and popcorn maize in comparison to the control conditions, whereas an increase was observed in sugar maize. In response to severe drought stress, HSP90 protein expression in dent maize increased gradually, although it remained below the levels observed under normal conditions. However, a notable decline was observed in popcorn and sugar maize (Fig. 5).

The aforementioned changes are also evident in plant morphology. As illustrated in Fig. 6, there was a notable shortened plant height, decrease in leaf area as the drought intensified, as well as a decline green color intensity due to the decrease in chlorophyll content.

Correlation analyses for studied parameters

The results of the two-tailed Pearson correlation analysis conducted to determine the relationships between the parameters examined in the study are presented in Table 2. In particular, there was a positive and highly significant correlation between HSP70 gene expression and PRO content, while there was a negative and significant correlation between HSP70 gene expression and APX activity. The expression of the HSP90 gene was found to be positively and significantly correlated with MD%, LOT, SOD, APX, and CAT activities. Conversely, it was negatively and significantly correlated with MDI, RWC, and PRO.