Physiological and biochemical effects of 24-Epibrassinolide on drought stress adaptation in maize (Zea mays L.)

Plant materials and growth conditions

For the present study, a field experiment under the rain-out-shelter facility at the ICAR-Indian Agricultural Research Institute, New Delhi, India was conducted in a randomized block design (RBD) using two Indian maize hybrids, viz. Vivek hybrid 9 (relatively drought-tolerant) and Bio 9637 (relatively drought-susceptible) during the kharif (monsoon) season in triplicate. Seeds of both hybrids were obtained from the Indian Institute of Maize Research, New Delhi, India. Three different treatments (T₀ = irrigated, T₁ = drought, T₂ = drought+EBR) were studied. Drought was imposed by withholding irrigation 2 weeks before anthesis and nearly 30 ml EBR (1 µM) per plant was applied on the same day, through the foliar spray using the handheld sprayer. The EBR concentration used for the present study was identified based on our preliminary seedling survival experiment conducted on Vivek hybrid 9. Flag leaves from three plants in each replication were collected at 7, 14, 21, and 28 days after foliar application of EBR.

Seedling survival

A seedling survival experiment was conducted on Vivek hybrid 9 to optimize the EBR concentration. A total of 100 seeds in three replications were sown in small pots (6 cm) under normal irrigation in the net house until the V3 growth stage (when the collar of 3^rd leaf from the bottom is visible), after which irrigation was withheld and five different concentrations of EBR viz., 0, 0.1, 0.5, 1.0, 1.5 and 2.0 µM, were foliar sprayed. Seedling survival rates were recorded at 24, 48, 72, 96, and 120 h after the withdrawal of irrigation.

Morphological parameters

Morphological parameters such as plant girth, leaf area index (LAI), and root biomass were recorded at 7, 14, 21, and 28 days after treatment (DAT) using three plants in each replication for both hybrids. Root biomass was measured using a non-destructive method using a root capacitance meter.

Physiological parameters

The relative water content (RWC) of the leaves was measured using the method described by Weatherly (1950). Leaf water potential was measured by using an HR-33T Dew Point Microvoltmeter (Wescor, Stoneham, MA, USA). Photosynthetic rate (A) was measured with an infrared gas analyzer (model LI 6400; LI-COR, Biosciences, Lincoln, NE, USA) equipped with an air supply unit and a broad leaf chamber (aperture area 6.25 cm²; 25–27 °C air temperature, CO₂ concentration at 550 ± 50 μL L⁻¹). The normalized difference vegetation index (NDVI) was recorded using a Green Seeker optical sensor (Trimble Navigation Ltd., Sunnvale, CA, USA). Anthesis-silking-interval (ASI) was measured as the number of days between when 50% of the plant population was at the anthesis stage (beginning of pollen shed from the tassel (male inflorescence)) to when 50% of the plant population was at silking stage (the first appearance of stigmas (silk) from the surrounding husk as the primary ear (female inflorescence)) (Bolanos & Edmeades, 1996). Leaf senescence was measured by scoring on a scale from 1 (100% green leaf area) to 9 (0% green leaf area) (Bhoite et al., 2019; Anderegg et al., 2020).

Biochemical parameters

Total soluble proteins were estimated using the method described by Bradford (1976). Protein was extracted, and the absorbance shift of the dye Coomassie Brilliant Blue G-250 added to the protein extract at 595 nm was recorded. Protein was calculated by the increase in absorbance, which is proportional to the amount of dye bound to the protein in the sample.

MSI was estimated according to the method described by Premchandra, Saneoka & Ogata (1990). In brief, leaf discs were cut from the midrib in the middle of the leaves and then rinsed with distilled, deionized water three times in a beaker. These discs were soaked for a day at 10 °C in 30 ml of 30% PEG 600 to undergo desiccation treatment. In contrast, control leaf discs were placed in equivalent amounts of deionized distilled water. Finally, the discs received three quick washes using deionized distilled water. Purified distilled water (30 ml) was added to the desiccated and control samples, and the leaf discs were stored at 10 °C for 24 h. Afterward, the flask was heated to 25 °C, and shaken thoroughly, and then the electrical conductivity was determined using an Electric conductivity meter. The leaf tissues were subjected to autoclaving for 15 min and reheated at 25 °C. The electrical conductivity was then measured again with three replicates each for both desiccation treatment (T) and control (C). Cell membrane stability of leaf tissues was determined by calculating the amount of damage/injury expressed as a percentage, using the following formula:

$$\%\text{\hspace{0.17em}injury=1-}({\text{1-T}}_{\text{1}}{\text{/T}}_{\text{2}})\text{/}({\text{1-C}}_{\text{1}}{\text{/C}}_{\text{2}})\times \text{100},$$

where T, and T2 = the first and second conductivity measurements of the desiccation treatment, respectively, and C, and C2 = the first and second conductivity measurements of control, respectively.

Proline was extracted and its concentration was determined by the procedure described by Bates, Waldren & Teare (1973). Leaf tissue was homogenized in 3% sulfosalicylic acid, and the homogenate was centrifuged and filtered through Whatman filter paper. Two milliliters of this filtrate were mixed with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid in a test tube. The mixture was heated at 100 °C in a water bath for 1 h and absorbance was recorded at 520 nm. Glycine betaine was estimated by recording the absorbance of the betaine-periodide complex formed with iodine in an acidic medium at 365 nm as described by Greive & Grattan (1983).

SOD activity was estimated by recording the decrease in optical density of formazone produced by superoxide radicals and nitro-blue tetrazolium dye by the enzyme. The absorbance was recorded at 560 nm, and one unit of enzyme activity was taken as that amount of enzyme, which reduced the absorbance reading to 50% in comparison with the tubes lacking enzyme (Dhindsa & Matowe, 1981). CAT assay was based on the absorbance of H₂O₂ at 240 nm. A decrease in absorbance was recorded over 1 min as described by Aebi (1984). The APX assay was based on absorbance by ascorbic acid at 290 nm in the UV range, and a decrease in absorbance due to the oxidation of ascorbic acid to mono-dehydroascorbic acid and dehydroascorbic acid (Nakano & Asada, 1981).

Yield and its attributes

The hybrids were grown in three rows of 3 m length with 75 cm row-to-row distance. Yield-related traits such as cob weight, cob girth, 100-grain weight, and Harvest Index (HI) were recorded after harvest using three plants from the middle row in each replication.

Statistical analysis

The data include means of three replicates along with standard deviations (SD). The fixed factors in this study include genotypes (hybrids), treatments, and days after treatments. R software version 3.6.1 (R Core Team, 2019) was used to analyze the data and subjected to a two-way analysis of variance (ANOVA). The means were compared between hybrids and among the treatments by HSD (Honest Significant Difference) at p < 0.05 level using Tukey’s test.

Plant survival at critical soil moisture levels

In our study, 2-week-old maize seedlings exposed to drought stress as well as treated with different concentrations of EBR exhibited a differential response. There was no effect of drought (withdrawal of irrigation) on seedling mortality up to 24 h when the soil moisture/water content was 15.8% (Fig. S1). However, with time, soil moisture depleted, which also affected seedlings’ survival. At 120 h after treatment, seedling survival ranged between 0.0% to 23.3% at soil moisture of 7.8%. The most effective concentration of EBR for seedling survival under drought stress was 1.0 µM (Fig. S1).

Plant growth characteristics

Plant girth increased in control plants, but a reverse pattern was observed under drought stress conditions up to 28 DAT. Plant girth was reduced by 2.67% and 12.41% in Vivek hybrid 9 and Bio9637, respectively (Table S1 and Fig. S2A). However, the EBR application did not significantly affect plant girth under drought stress in either hybrids. The Leaf area index increased in control plants but decreased under drought stress in both hybrids. A decrease of 6.59% and 10.99% was observed in Vivek hybrid 9 and Bio 9637, respectively, under drought compared with the control (Table S1 and Fig. S2B). There was no significant difference in LAI between the two hybrids for up to 21 DAT. EBR applications significantly improved LAI in both hybrids under drought stress. Root biomass increased up to 21 DAT and thereafter decreased in both hybrids in control plants. A steady decrease in root biomass was observed in both hybrids under drought stress, but the effect was more pronounced in Bio 9637. A reduction of 14.54% and 23.03% was recorded in Vivek hybrid 9 and Bio 9637, respectively, under drought stress. EBR application improved significantly root fresh biomass under drought stress (Table S2 and Fig. S3A). Leaf senescence score (1–9 scale) increased with the age of the plant in both hybrids and drought stress further enhanced it drastically. In Bio 9637, drought caused a nearly 3-fold increase in leaf senescence score. EBR helped in decreasing the leaf senescence score by 27% in Bio 9637. However, there was no significant difference in leaf senescence with respect to the interaction between genotypes/hybrids and treatment (Fig. 1 and Table S3). NDVI, which is an indicator of the greenness and health status of the plant, decreased with the age of the plant for all the treatments in Vivek hybrid 9, whereas, in Bio 9637, NDVI in the control increased up to 7 DAT. Drought treatment caused around 11–13% decrease in NDVI in both hybrids. EBR application caused a 16% increase in NDVI in Bio 9637, whereas, in Vivek hybrid 9, it caused only 1% increase in NDVI compared with untreated plants under drought (Fig. 1 and Table S3). ASI of both hybrids increased under drought conditions while EBR application restricted the increase in ASI in both hybrids. However, the increase in ASI was much higher in Bio 9637 than in Vivek hybrid 9 under drought stress (Fig. S4).

Photosynthetic rate

With increasing plant age, the rate of photosynthesis was reduced in both hybrids in all three treatments. (Fig. 1 and Table S3). Under drought conditions, Bio 9637 showed a significantly higher decrease in the photosynthesis rate than Vivek hybrid 9. The decrease in photosynthesis was significant between treatments, genotypes, and their interaction (G×T) for all observations. EBR application improved the photosynthetic rate by 8.67% and 27.20% in Vivek hybrid 9 and Bio 9637, respectively, under drought stress (Table S3).

Plant water relations and membrane stability

Leaf RWC decreased under drought conditions in both hybrids up to 28 DAT. Significant differences in RWC were observed between genotypes and among the various treatments, however, their interaction effect (G×T) was non-significant, except at 14 DAT (anthesis stage). EBR application significantly improved leaf RWC in both the hybrids under drought stress (Table S2 and Fig. S3B). In the case of the leaf water potential trait, no significant difference was observed between treatment, between genotypes, and their interaction (G×T) on 7 DAT, whereas after that leaf water potential decreased drastically in Bio 9637, and this decrease was maximum at 28 DAT i.e. 35%. Leaf water potential after 4 weeks of drought decreased by 12% and 28% in Vivek hybrid 9 and Bio 9637, respectively. EBR application caused a 14% increase in leaf water potential in Bio 9637 (Table S2 and Fig. S3C). Similarly, the membrane stability index in both hybrids under drought conditions was reduced but sustained when EBR was applied under drought conditions. It was improved by 12% in Vivek Hybrid 9 and by 19% in Bio 9637 (Fig. 2 and Table S4).

Total soluble protein content and Osmolyte adjustment

There was a decrease in protein content under drought in both hybrids, but the decrease was more prominent in Bio 9637, with 38% lower protein content than the control. Whereas EBR treatment increased protein content in Bio 9637 by 52% compared with untreated plants under drought stress. However, Vivek hybrid 9 showed only a 10% decrease in protein content compared with untreated plants under drought treatment. In addition, there was no significant difference between hybrids at 14 DAT (Fig. 2 and Table S4). The proline content in both hybrids increased under drought conditions and after EBR application. The increase in Bio 9637 was more pronounced with EBR application as compared to untreated drought. It increased up to ~45% as compared to drought conditions. However, in the case of Vivek Hybrid 9, the proline content varied marginally when EBR was applied compared with drought alone (Table S5 and Fig. S5A). The glycine betaine content increased up to 28 DAT in both hybrids under all treatments. Under drought stress, an increase in glycine betaine content to the magnitude of 120% and 52% was observed in Vivek hybrid 9 and Bio 9637, respectively. EBR application further enhanced glycine betaine content under drought conditions. However, the effect was more pronounced in Bio 9637 (Table S5 and Fig. S5B).

Antioxidant enzyme activities

SOD activity increased in both hybrids under drought stress. A significant difference between treatments, genotypes, and G×T was observed in terms of SOD activity. EBR application increased the SOD activity by 15% and 32% in Vivek hybrid 9 and Bio 9637, respectively (Fig. 3 and Table S6). Similarly, a 74% increase in CAT activity in Vivek hybrid 9 under drought conditions was observed compared with the control, and activity was further enhanced by 23% upon EBR treatment. Bio 9637 also showed a higher catalase activity in drought conditions, but there was less increase than Vivek hybrid 9. Further, EBR treatment increased the CAT activity by 56% in the Bio 9637 hybrid. Thus, there was a significant difference in the CAT activity between treatments, between hybrids, and their interaction (Fig. 3 and Table S6). Furthermore, Vivek hybrid 9 showed 45% higher APX activity in drought conditions, and activity was further increased upon EBR treatment. Similarly, APX activity also increased in Bio 9637 under drought conditions by 23%. The EBR treatment was very effective in enhancing APX activity in Bio 9637, where it increased by 45% compared with drought-alone conditions (Fig. 3 and Table S6).

Yield attributes

Cob weight decreased in both hybrids under drought conditions. A reduction of 24% and 23% in cob weight was observed in the Vivek Hybrid 9 and Bio 9637 genotypes, respectively. The application of EBR improved cob weight in both hybrids under drought conditions. However, the interaction effect (G×T) was not significant (Table S7 and Fig. S6). Similarly, cob girth decreased in both hybrids under drought conditions. In Bio 9637, girth was decreased by 19%, whereas in Vivek Hybrid 9 it was reduced by 15%. Application of EBR showed improvement in cob girth under drought stress in both hybrids, but the effect was more pronounced in Bio 9637 (Table S7 and Fig. S6). Drought stress also negatively affects both hybrids. The 100-grain weight of both hybrids under drought but EBR application improved grain weight. The harvest index (HI) of both hybrids was reduced under drought conditions. It was reduced by 25% in Vivek Hybrid 9 and by 42% in Bio 9637. EBR application improved HI in both hybrids under drought-stress conditions. Significant differences were observed between hybrids and among treatments for HI. The interaction effect (G×T) was also found to be significant (Table S7 and Fig. S6).