|Phyton-International Journal of Experimental Botany|
Foliar Application of Cytokinin Modulates Gas Exchange Features, Water Relation and Biochemical Responses to Improve Growth Performance of Maize under Drought Stress
1Department of Agronomy, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, 1706, Bangladesh
2Department of Crop Botany, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, 1706, Bangladesh
3Department of Biochemistry and Molecular Biology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, 1706, Bangladesh
4Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Science, Texas Tech University, Lubbock, TX 79409, USA
*Corresponding Authors: Mohammad Golam Mostofa. Email: firstname.lastname@example.org; email@example.com; M. Rafiqul Islam. Email: firstname.lastname@example.org
Received: 27 June 2021; Accepted: 30 August 2021
Abstract: Improvement of plant performance under drought stress is crucial to sustaining agricultural productivity. The current study investigated the ameliorative effects of foliar-applied kinetin, an adenine-type cytokinin (CK), on growth and gas exchange parameters, water relations and biochemical attributes of maize plants under drought stress. Eighteen-day-old maize plants were subjected to drought by maintaining soil moisture content at 25% field capacity for 8 days followed by foliar application of kinetin at 0, 75, 150 and 225 mg L−1 (CK0, CK75, CK150 and CK225, respectively) to the plants for two-times at the 9-day interval. Results revealed that drought stress markedly reduced stem diameter, dry weight, chlorophyll content, gas exchange parameters and water balance but increased proline, malondialdehyde and soluble sugar contents, electrolyte leakage and senescence in maize leaves. Application of exogenous CK remarkably improved maize performance by modulating growth, gas exchange- and water relation-related parameters in a dose-dependent manner under drought stress. CK225 increased chlorophyll content (by 61.54%), relative water content (by 49.14%), net photosynthesis rate (by 39.94%) and transpiration rate (by 121.36%) and also delayed leaf senescence but decreased internal CO2 concentration (by 7.38%), water saturation deficit (by 40.40%) and water uptake capacity (by 42.49%) in both well-watered and drought-stressed plants. Nevertheless, CK application considerably decreased electrolyte leakage, proline, malondialdehyde and soluble sugar levels in drought-stressed maize plants, as also supported by heatmap and cluster analyses. Taken together, exogenous CK at proper concentration (225 mg L−1) successfully improved maize performance under drought conditions, thereby suggesting CK application as a useful approach to alleviate drought-induced adverse effects in maize plants, and perhaps in other important crop plants.
Keywords: Cytokinin; drought; maize; osmoprotection; photosynthesis; water-use-efficiency
Maize (Zea mays) is a promising cereal crop for achieving global food security . Drought, an intricate abiotic stress, is a major constraint for the intensification and sustainability of maize production worldwide . It is estimated that a single episode of drought costed 7% global yield loss in maize, which is predicted to be increased in the future due to climate change . In South Asia, most of the agricultural lands (65%) are frequently exposed to differential degrees of periodic drought, which is responsible for 10–15% yield loss every year in this region [4,5]. In the southwestern and northwestern regions of Bangladesh, 51% (17,243 km2) areas are extremely vulnerable to drought . The simulated models estimated an increase in yield loss from 9.0 to 12.0% and 5.6% to 6.3% for wheat and maize, respectively, by the end of the 21st century . However, maize is more sensitive to drought than wheat, particularly during the reproductive stage . Thus, finding effective mitigation strategies, including sustainable adaptation practices are required to address drought effects on maize growth and productivity.
Drought negatively affects plant growth and development, resulting in yield loss to a number of crops, including maize [8,9]. Drought stress causes abnormal changes in morphology, water status, gas exchange, and chlorophyll content in plants [10,11]. Plants also respond to water shortage by accommodating various physiological, biochemical and molecular changes, leading to adaptation to drought stress . Currently, there are hardly any economically viable technologies available to facilitate maize production under water-shortage conditions. However, agronomic manipulations, including the application of exogenous plant growth regulators are considered promising approaches for improving maize growth and development, thereby contributing to sustainable maize production under drought-stressed environments [13,14].
Drought can adversely affect root and shoot growth by disrupting endogenous levels of phytohormones . Thus, maintaining hormonal balance is crucial to support plant growth under adverse environmental conditions. A wealth of studies demonstrated that application of exogenous phytohormones, including cytokinin (CK) boosted plant tolerance to drought stress by modulating numerous physiological and biochemical functions of plants . Cytokinins (CKs) are multifunctional phytohormones that control cell proliferation and vascular development, root function and nodulation, and promote apical dominance, all of which govern plant growth processes under both normal and stressed situations [7,17–19]. Being a growth regulator, CK is involved in several fundamental aspects of plant development and stress tolerance [20,21]. Application of exogenous CK improved plant growth by regulating many key processes of plant like cell division and expansion [22–24] photosynthesis and stomatal behavior [25,26], biogenesis of chloroplasts and senescence [27,28], water exchange and ion uptake , formation and protection of cellular structures , the induction and activation of osmoprotectants and antioxidant systems . The positive effects of CK application have been reported in many plant species, including wheat (Triticum aestivum), barley (Hordeum vulgare), mungbean (Vigna radiata) and faba bean (Vicia faba) under different environmental stress conditions [32–35]. However, the putative roles of CK in alleviating drought stress in maize were not thoroughly investigated.
In the current study, we investigated the beneficial roles of CK in the improvement of maize performance under drought stress conditions. For this purpose, we evaluated CK-mediated drought tolerance mechanisms at the physiological and biochemical levels by analyzing several growth-related parameters, gas exchange properties, osmoprotection, water status and membrane damage in maize. We also searched for the effective concentration of CK needed for enhanced maize performance under greenhouse settings with restricted water supply.
2 Materials and Methods
2.1 Experimental Site, Plant Material and Plant Growth Conditions
The experiment was conducted under semi-controlled environmental condition in the Stress Research Site of the Department of Agronomy, Bangabandhu Sheikh Mujibur Rahman Agricultural University (90.26° E and 24.09° N), Gazipur, Bangladesh during the period from July to September 2014 using a widely used variety BARI maize-5 developed by Bangladesh Agricultural Research Institute (BARI). A total of 48 wagoner pots (25 cm diameter and 30 cm height) were taken to carry out the study. Soil used in the experiment was sandy clay loam that attained the full field capacity at 27.8% volumetric water content. Three seeds of maize were sown directly in plastic pots containing 12 kg of soil and organic compost (3:1, v/v) under the conditions of 12.3/11.7 h light/dark period. Chemical fertilizers, including urea, triple super phosphate, muriate of potash and gypsum were applied as per recommendation of BARC . Most of the seedlings emerged within 5–6 days of sowing. The pots were thinned to one healthy plant per pot at 8 days after emergence (DAE). Two sets of pots of which Set-1 containing 24 plants was selected for drought treatments, whereas Set-2 containing 24 plants was kept for well-watered (WW) treatments.
2.2 Imposition of Drought and Hormonal Treatments
Both sets of pots were properly irrigated to ensure better growth of the plants. Eighteen-day-old plants of Set-1 were subjected to drought by withdrawing regular irrigation and then maintaining 25% field capacity (approximately 7% volumetric moisture content) for a period of 8 days. On the other hand, the soil moisture contents of Set-2 pots containing WW plants were maintained at full field capacity (approximately 27.8% volumetric moisture content). The soil moisture levels at 15 cm depth of all the experimental pots were carefully monitored every day using a digital soil moisture meter (PMS-714, Lutron Electronic Enterprise Co., Ltd., Taiwan) throughout the experimental period. Kinetin, an adenine-type CK, was used as an exogenous source of CK. After the drought period, both drought-stressed and WW plants were foliar-sprayed with different concentrations of kinetin (0, 75, 150 and 225 mg L−1, hereafter defined as CK0, CK75, CK125 and CK225, respectively) at 26 DAE and 35 DAE. The experiment consisted of eight treatments, including (i) control (well-watered, WW) (CK0), (ii) WW + 75 mg L−1 kinetin (CK75), (iii) WW + 150 mg L−1 kinetin (CK150); (iv) WW + 225 mg L−1 kinetin (CK225); (v) drought stress (DS); (vi) DS + CK75; (vii) DS + CK150 and (viii) DS + CK225. All the treated and untreated plants were allowed to grow until final harvest at 51 DAE for determinations of dry weight, gas exchange parameters, leaf chlorophyll contents, water status and biochemical parameters. The experiment was laid out in a randomized complete block design with six replications (n = 6) and each replicate included one plant (i.e., 6 plants per treatment). There was total 48 pots (6 pots × 8 treatments = 48) in the current study.
2.3 Measurement of Growth Parameters, Gas Exchange Properties and Water Status
Growth related parameters, such as stem diameter, leaf senescence, fresh weight (FW), dry weight (DW) of different plant parts, including roots, stems and leaves were determined following the procedure reported by Akter et al.  and Borras et al. . Leaf gas exchange parameters like net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr) and intercellular CO2 concentration (Ci) were recorded in fully developed and expanded third leaf from the top of each plant at 11.30 am to 12.30 pm of the local time using the portable photosynthesis measurement system Li 6400XT (Model-Li 6400XT, LI-COR Inc., Lincoln, NE, USA). Water relation traits, including leaf relative water content (RWC), and water saturation deficit (WSD) and water uptake capacity (WUC) were measured in maize leaves following the protocols reported by Schonfeld et al.  and Sangakkara et al. , respectively. The RWC, WSD and WUC were calculated based on the following formulae: RWC (%) = [(FW – DW)/(TW–DW)] × 100; WSD (%) = [(TW – FW)/(TW – DW)] × 100; WUC = (TW – FW)/DW, where, FW, fresh weight; DW, dry weight and TW, turgid weight [38,39].
2.4 Estimation of Total Chlorophyll Content in Maize Leaves
Total chlorophyll contents in the leaf samples were measured following the procedure of Witharn et al.  by using double beam spectrophotometer (Model 200–20, Hitachi, Japan). Twenty mg of leaf sample was collected in a bottle containing 20 mL acetone and kept in bottle for 48 h. The absorbance of the supernatants was spectrophotometrically read at 645 and 663 nm using 80% acetone as blank. Total chlorophyll content was calculated using the formula adopted by Witharn et al. .
2.5 Determination of Proline and Total Soluble Sugar Contents in Maize Leaves
Proline content was determined following the procedure of Bates et al. . Briefly, fresh leaf samples (0.5 g) were crushed in 3% sulfosalicylic acid solution and allowed to settle for 5 min. After centrifugation at 15,500 g, 2 mL of filtrate was mixed with 2 mL each of acid ninhydrin glacial acetic acid solution. The mixture was heat at 100 °C for 60 min followed by cooling on ice bath for 30 min. Toluene was used to collect the chromophore, and the absorbance of the collected solution was read at 520 nm using toluene as blank. Proline content was determined from a standard graph developed with a series of proline concentrations. Total soluble sugar content was determined using anthrone reagent following the procedure developed by Kirankumari et al. .
2.6 Measurement of MDA and Electrolyte Leakage in Maize Leaves
The level of MDA was determined using thiobarbituric acid (TBA)-dependent method of Health et al. . MDA content in leaf samples was calculated using an extinction coefficient of 155 m M−1 cm−1. Drought injury index or electrolytic leakage (EL) was tested following the method reported by Kocheva et al. . Briefly, 20 leaf discs (size of the disc) of drought-stressed and -unstressed plants were washed with distilled water to clean the disc. The disc samples were then immersed in 20 mL distilled water at room temperature. After 24 h, the conductivity of the solutions (Lt) was read using a conductivity meter. The samples were autoclaved at 120 °C for 15 min followed by cooling at room temperature for 20 min. The conductivity of the resultant solutions was read again (Lo). The EL was calculated using the following formula: EL (%) = (Lt/Lo) × 100.
2.7 Statistical Analysis
Analysis of variance was conducted using MSTAT-C software  and significant difference among the means was compared by post-hoc least significant difference (LSD) test at P < 0.05. Heatmap and cluster analysis were carried out using R-4.1.0 for win (http://CRAN.R-project.org/) (accessed on 25 June 2021). Trait mean values were normalized and the library pheatmap was adapted for generating heatmap and hierarchical clusters (distance = Euclidean and method = ward. D2 .
3.1 Effects of CK on Growth Associated Attributes of Maize under Drought Stress
Growth parameters, such as, DW of root, stem and leaf were determined to evaluate the effects of CK in the alleviation of drought effects on maize growth and development. Drought stress negatively affected plant biomass by reducing root DW (74.21%), stem DW (81.90) and leaf DW (69.69%) in comparison with those observed in maize plants under WW conditions (Table 1). However, the application of exogenous CK significantly increased the dry biomass of root, stem and leaf in both control and drought-stressed plants when contrasted with their respective controls. Particularly, application of CK225 to drought exposed maize plants increased root DW, stem DW and leaf DW by 52.00%, 71.59% and 73.00%, respectively, relative to drought-stressed only plants (Table 1). Under non-stressed conditions, CK225 also increased root DW, stem DW and leaf DW by 15.67%, 24.12% and 29.68%, respectively, in control plants. Interestingly, dry biomass accumulation increased with the increase of CK concentration and CK225 showed the most effective roles in counteracting the adverse effects of drought on maize growth (Table 1).
Plants were exposed to drought stress at 26 DAE and continued up to 51 DAE (harvesting time) along with different concentrations of kinetin sprayed at 26 DAE and 35 DAE. Means followed by different alphabetical letter(s) within a column are significantly different from each other at P < 0.05 according to Tukey’s post hoc test. CK75, CK150 and CK225 represent 75, 150 and 225 mg L−1 cytokinin, respectively. DAE, days after emergence; DS, drought stress; DW, dry weight; WW, well-watered.
3.2 Effects of CK on Stem Diameter and Leaf Senescence of Maize under Drought Stress
Stem diameter and leaf senescence were determined at 23, 30, 37, 44 and 51 DAE to evaluate the effects of CK before and after drought stress on these two parameters (Figs. 1 and 2). Drought stressed plants showed a reduction of stem diameter by 12.10% to 30.90% but an increase of leaf senescence by 17.10% to 34.80% from 23 DAE to 51 DAE compared with WW plants. Accordingly, application of CK increased stem diameter but delayed leaf senescence in drought-stressed maize to some extent in an age-dependent manner (Figs. 1 and 2). The percent reduction of stem diameter in drought-stressed plants was 5.6% to 24.7% relative to control plants upon application of CK (Fig. 1). Moreover, CK also increased stem diameter of WW plants by 0.8% to 8.8% relative to CK-devoid control plants (Fig. 1). In case of leaf senescence, we recorded that CK225 in non-stressed WW plants had the lowest leaf senescence rate (48.6%) relative to control at 30 DAE (Fig. 2). However, CK150 and CK225 delayed the leaf senescence under drought stress condition, and the percent relative change was −23.1% to −37.8% as compared with WW plants (Fig. 2). Among different concentrations, CK225 was proved to be more efficient to reduce the effects of drought on both stem diameter and leaf senescence.
3.3 Effects of CK on Gas Exchange Parameters in Maize under Drought Stress
Net photosynthesis rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were recorded at 51 DAE to assess the effects of CK on these gas exchange characteristics of maize plants (Fig. 3). Exposure of maize plants to drought stress resulted in a significant reduction in photosynthetic attributes, including Pn, gs, and Tr by 53.59%, 42.27%, and 85.12%, respectively, whereas increment of Ci was observed by 12.24% when compared with WW plants. The application of exogenous CK in both control and drought-stressed plants improved the gas exchange attributes excluding Ci (Fig. 3). The higher the concentrations of CK, the more enhancements in gas exchange characteristics were recorded. The CK-application at CK225 following drought exposure resulted in an improvement of Pn, gs, and Tr by 39.94%, 27.73% and 121.36%, respectively, and a reduction of Ci by 7.38% in comparison with those observed in drought-stressed only plants. This result indicated that CK at the higher dose (225 mg L−1) proved to be more effective than those of lower doses (75 and 150 mg L−1) in improving gas exchange parameters under both normal and drought stress conditions (Fig. 3).
3.4 Effects of CK on Water Status in Maize Plants under Drought Stress
The effects of CK on plant water status in terms of relative water content (RWC), water saturation deficit (WSD), water uptake capacity (WUC), and electrolytic leakage (EL) were documented at 51 DAE in maize plants (Table 2). Drought-stressed plants showed a significant decrease in RWC (by 35.62%) but an increase in SWD (by 83.36%), WUC (by 135.34%) and EL (by 29.69%) when compared with control plants. The exogenously applied CK in both control and drought-stress plants improved plant water status, but the degree of improvement was much higher in drought-stressed plants (Table 2). Besides, the higher the concentrations of CK, the more augmentations in plant water status were observed in control and drought-stressed plants. The application of CK at the higher dose (CK225) resulted in much improvement of RWC by 49.14%, but reduction of WSD, WUC, and EL by 40.40%, 42.49% and 33.68%, respectively in drought-stressed plants when compared with drought-stressed only plants. Our data also showed that CK225 was more effective than CK75 and CK150 in improving plant water status under both normal and drought stress conditions (Table 2).
Plants were exposed to drought stress at 26 DAE and continued up to 51 DAE (harvesting time) along with different concentrations of kinetin sprayed at 26 DAE and 35 DAE. Means followed by different alphabetical letter(s) within a column are significantly different from each other at P < 0.05 according to Tukey’s post hoc test. CK75, CK150 and CK225 represent 75, 150 and 225 mg L−1 kinetin, respectively. DAE, days after emergence.
3.5 Effects of CK on Biochemical Status in Maize Plants under Drought Stress
The effects of CK on the proline, soluble sugars, malondialdehyde (MDA), and chlorophyll in the leaves of maize plants were observed at 51 DAE (Fig. 4). The drought-stressed maize plants experienced much elevation in proline (by 137.58%), soluble sugars (by 73.95%), and MDA (by 55.69%) but a decline in total chlorophyll contents (by 56.41%) compared with control plants. Both drought-stressed and control plants applied with CK displayed a reduction in proline, soluble sugar and MDA contents but an increase in total chlorophyll content. Such decreasing rate of proline, soluble sugar, and MDA and increasing rate of chlorophyll contents was much higher in drought-stressed plants if compared with control plants. Interestingly, a high concentration of CK (CK225) had more ameliorating effects by reducing the accumulations of proline, soluble sugar, and MDA and increasing chlorophyll content in both drought-stressed and control plants (Fig. 4). Exogenous application of CK225 to drought-stressed maize plants reduced proline, soluble sugars and MDA contents by 47.89%, 40.25%, and 27.68%, respectively, relative to drought-stressed only plants. Under non-stressed conditions, CK225 reduced the levels of proline, soluble sugars and MDA by 17.61%, 22.04% and 9.81%, respectively, and increased total chlorophyll contents by 61.54% in comparison with control plants. Results indicated that CK at a higher dose (CK225) proved to be more effective than those of lower doses (CK75 and CK150) in positively modulating the biochemical parameters of maize plants under both normal and drought-stressed conditions (Fig. 4).
3.6 Comparative Responses Across Treatments
Comparative heatmap analysis revealed two distinct clusters among parameters measured in this study (Fig. 5). Cluster 1 consisted of dry weights, Pn, Gs, Tr, RWC, total chlorophyll contents and stem diameter. Proline content, Ci, WUC, MDA, WSD, TSS, EL and leaf senescence were placed in Cluster 2. At the treatment levels, well-watered maize plants foliar sprayed with different doses of CK were clustered in the Group 1, while except CK225, drought-stressed maize plants with or without foliar application of CK placed in Group 2 (Fig. 5). However, DS + CK225 displayed significantly higher amelioration effects on drought stress, indicated by comparatively lower contribution of the traits of Cluster 2 and increase in the RWC and stem diameter and higher retention of chlorophyll contents assembled in the distinct Group 3 (Fig. 5).
Drought, the most prominent threat to maize production, accelerates leaf senescence and chlorosis, leading to a decrease in photosynthesis, biomass production and yield potential of plants [47,48]. The exogenously applied CK is known to regulate plant growth and development by controlling several plant processes, including photosynthesis and osmoprotection, oxidative stress protection during drought-succession of plants [49,50]. The current study aimed at assessing the effectiveness of CK in alleviating drought adversities in commercially vital crop maize.
The benefits of foliar application of CK on stem diameter, leaf chlorophyll content, delay of leaf senescence, and greater photosynthesis rate were positively corelated with the biomass production in maize plants under drought stress conditions (Table 1; Figs. 1–3), as also observed by Akter et al. . Application of exogenous CK might have enhanced the endogenous levels of CK, which later stimulated the overall performance of maize plants to overcome water-shortage caused negative effects on growth (Table 1). The CK-mediated enhancement of stem diameter attributed to the promotion of cell division . The current study showed that CK application acted positively in reduction of leaf senescence and chlorophyll loss (Figs. 2 and 4). The reduction in chlorophyll concentration, damage of the photosynthetic machinery, and oxidative damage are all well-known consequences of natural or stress-induced leaf senescence . Drought causes early leaf and flower senescence in plants by increasing the production of senescence-associated genes that code for cysteine proteases . CK has been proved to be beneficial for drought adaptation of plants by delaying the leaf senescence, preventing protein breakdown, activating protein synthesis, and assembling nutrients from nearby tissues . Liu et al.  reported that exogenously administered CK controlled rice flag leaf aging via regulating chlorophyll degradation, membrane deterioration, and senescence-associated gene expression.
Drought stress significantly depressed canopy and leaf gas exchanges to a varying degree, showing a greater reduction in Pn, gs and Tr of the maize plants (Fig. 3). These results are in agreement with the reports of Vitale et al. , Naeem et al.  and Ashraf et al. , who demonstrated that photosynthesis of drought stressed plants appeared to be reduced primarily because of stomatal closure and later by a reduction in photosynthetic capacity. High stomatal conductance is the prerequisite for an increase in CO2 fixation per unit of leaf area to allow a greater photosynthesis rate . Under drought circumstances, the stomata remain closed for an extended length of time, reducing CO2 absorption and water loss to preserve plant water status [60,61]. The exogenous application of CK increased Pn, gs and Tr in maize plants grown under normal or drought stress conditions (Fig. 3), which is in conformity with the findings of Wu et al.  in eggplant under salinity stress. In the current study, we observed an increased Tr and gs in parallel with increased RWC in maize plants treated with CK. Our results suggest that CK helped maize plants to maintain an elevated Tr for enhancing Pn to support plant growth under water-shortage conditions. It is likely that CK did not play roles in water saving through the reduction of Tr in maize plants exposed to water-limited stress.
It is worth noting that understanding the current state of water relations is critical for better crop management in drought-prone locations. Among several water-related parameters, RWC is an important characteristic that used as an integrative indicator for dehydration tolerance [62–64]. A decrease in RWC due to drought resulted in a loss of turgor, which caused limited water availability for the cell division process in plants [65,66]. In this study, we observed that leaf RWC decreased during drought stress, but CK-treated plants showed a comparatively higher leaf RWC under both normal and water-limited conditions (Table 2), which supported the results of Chang et al. . We also noticed that RWC gradually increased upon increasing the doses of CK in drought-affected maize plants (Table 2). This result suggested that CK positively regulated water status in maize plants to cope with the adverse effects of drought. The observed high WSD in drought-stressed maize plants indicated that drought stress caused a greater degree of water deficit, which corroborated the findings of Islam et al.  in mung bean, Chowdhury et al.  in French bean and Mahmud et al.  in potato. On the other hand, foliar application of CK greatly decreased the WSD in both well-watered and drought stress conditions (Table 2), indicating that CK application helped maize plants to preserve water status for better growth performance. WSD is inversely correlated with the RWC in leaves , which could be resulted from the adjustment of physiological traits like root structure and root density. It has been reported that shoot-driven CK basipetally moved towards the roots, leading to the enlargement of the root system for foraging more area to absorb water under water-shortage conditions . Thus, CK-induced modulation of root structure and growth can help plants maintain better water status to thrive under water deficit conditions . However, WUC of drought-stressed plants was greater than that of control plants, suggesting that maize plants tried to compensate water deficiency by increasing WUC. On the contrary, application of CK reduced WUC in both control and drought-stressed plants (Table 2), implying that CK is more able to maintain internal water status rather than increasing WUC in maize plants.
Plants respond to drought stress by adopting different physiological and biochemical strategies. Plants accumulate various osmoprotectants to protect themselves from mild to severe water-deficit stress. Among the osmoprotectants, proline and soluble sugars are the two most widely studied organic solutes because of their considerable importance in conferring stress tolerance in plants under water-shortage conditions [73,74]. It is well documented that accumulation of proline acts not only in osmotic adjustment, but also participate in oxidative stress protection in plants [75–77]. Proline can also supply energy for growth and survival of plants, particularly under stressful conditions . Thus, accumulation of proline refers to an important adaptation strategy of plants to overcome deleterious effects of abiotic stresses, including drought [78,79]. In the current study, drought stress in maize plants resulted in a considerable proline buildup (Fig. 4A), as was also documented in other plant species, including wheat and soybean facing water shortages [12,80]. However, foliar application of CK reduced proline content in the leaves of maize plants, suggesting that CK application did not require the accumulation of much proline to manage drought stress. It is likely that CK supplementation eased maize suffering from negative effects of drought, which resulted in decreased accumulation of proline. Indeed, proline is used as stress indicator, and its accumulation also corelated with the intensity of stress generated in plants under abiotic stress conditions, including drought [12,81,82]. Our results are in parallel with the earlier findings of Das et al. , who observed reduced accumulation of proline in CK-sprayed NaCl-stressed plants. We also observed significant increment of soluble sugar contents in drought-stressed maize plants (Fig. 4B), indicating that soluble sugars functioned in osmotic stress management under drought conditions . However, application of CK lowered the accumulation of soluble sugars in a concentration dependent manner under drought stress conditions. This result was in agreement with the findings of Agarwal et al.  in callus cultures of cowpea and Yadav et al.  in Cicer, where they found kinetin as an osmotic adjustment stimulator. It has been reported that CK-overproducing transgenic rice PSARK::IPT showed higher hexokinase activity and lower levels of soluble sugars like glucose and fructose than wild-type under water deficit conditions . It is plausible that increased levels of CK rendered enhanced glycolysis of carbohydrates, thus depletion of soluble sugars, to supply adequate amounts of metabolites for facilitating high energy productions in response to drought.
Malondialdehyde is commonly used as an indicator of oxidative stress in plants under adverse environmental conditions. Lower MDA level indicates the higher antioxidant capacity in plants, thereby reflecting higher drought resistance as suggested by Izabela et al.  and Weng et al. . In this study, MDA content significantly increased under drought condition in maize plant (Fig. 4C), which is in agreement with the findings of Sarafraz-Ardakani et al. . According to their findings, MDA content was increased by 503% and 750% in drought-tolerant and drought-sensitive wheat cultivars, respectively. We observed that CK treatment lowered MDA contents in the leaves of maize plants in a concentration-dependent manner, and CK at 225 mg L−1 exhibited best performance in lowering MDA level. Heatmap analysis also showed that CK225 mitigated drought-stress principally by lowering MDA content, reducing electrolyte leakage and increasing RWC in the leaves of maize plant (Fig. 5). Current findings are in agreement with earlier report on creeping bent grass . Our results suggest that CK might play an indirect role in the maintenance of antioxidant systems, which protected maize plants from drought-induced oxidative damages . Indeed, EL also significantly decreased upon application of CK to the drought-stressed maize plants (Table 2). These results suggest that CK supplementation was effective in reducing the membrane damage. The CK-mediated recovery from membrane injury and increase in cell viability was reported in wheat plants under PEG-induced drought stress conditions .
Drought stress resulted in a reduction of morpho-physiological attributes and growth performance of maize plants at the early developmental stage. On the other hand, application of exogenous CK to drought-exposed maize plants caused an improvement of growth, which was positively associated with better gas exchange and photosynthesis parameters, and water relation characters. The reduced levels of stress indicators like proline, MDA and electrolyte leakage upon supplementation of CK indicated that CK had a soothing effect in overcoming drought effects on maize plants. Taking together, CK application improved drought tolerance in maize, and CK at 225 mg L−1 (CK225) was shown to be the most beneficial in alleviating drought effects on maize plants. However, a further study is required to determine the precise dose of CK and mechanistic insight of CK-mediated drought tolerance in maize under field conditions.
Author Contributions: M.R.I. designed and supervised the experiment. M.S.I. performed the experiment, harvested plant materials and collected field data. N.A. oversaw experiment, analyzed data and drafted manuscript with input from M.R.I and M.G.M. M.M. performed biochemical analysis and analyzed data. M.R.I. and M.G.M wrote, revised and finalized the manuscript.
Funding Statement: This research work was supported by the Ministry of Science and Technology, Government of Bangladesh. We also thank Bangladesh Agricultural Research Institute (BARI) for kindly providing the maize seeds for conducting the experiment.
Conflicts of Interest: The authors declare that they have no conflicts of interest.
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