Insight into 5-aminolevulinic acid-induced modulation of cellular antioxidant metabolism to confer salinity and drought tolerance in maize

The current study investigated the comparative oxidative damage in twomaize seedlings induced by saline, drought, and combined stress and the ameliorative role of two different doses (20 and 80 μM) of 5-aminolevulinic acid (ALA) against the above-mentioned stresses. Hydroponically grown 10-day-old maize (Zea mays, var. BARI Hybrid Maize-7 (BHM-7) and BARI Hybrid Maize-9 (BHM-9)) seedlings were exposed to 12 dS/m of saline solution, 200 mM mannitol-induced drought stress alone and their combined stress for 7 days. Result revealed that individual stresses retard the plant growth to some degrees; however, their combined stress has more detrimental effects, which might be correlated with lipid peroxidation (MDA)-induced oxidative stress in seedlings, enhanced Na/K ratio, and augmented generation of superoxide (O2 ) and hydrogen peroxide (H2O2). In contrast, exogenous ALA supplementation at 20 μM concentration markedly recovered from chlorosis and growth inhibition, substantially scavenged reactive oxygen species (ROS) and MDA by preserving ionhomeostasis and relaxing oxidative stress; also, by boosting catalase (CAT) and glutathione S-transferase (GST), and exclusively via depressing the activity of lipoxygenase (LOX) antioxidant enzyme. On the contrary, 80 μM ALA made things worse; nevertheless, higher activities shown by other antioxidant enzymes; like, superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), and glutathione peroxidase (GPX), which were related to lessen the oxidative damage by highly produced O2 •− and H2O2 under combined stress. Non-denaturing gel electrophoresis was done for further confirmation. However, ALA importantly increased the photosynthetic pigment contents in both genotypes irrespective of doses. Nevertheless, GST might have assisted the plants to escape from the herbicidal effect by detoxification. However, in the combined stress condition, high ALA concentration may have some positive role to play. Our findings also showed that BHM-9 performed better than BHM-7. Therefore, ALA at lower concentration was effective for single stress of saline and drought, while higher concentration can improve plant survival under combined stress.


Introduction
Ever-changing climatic conditions are intimidating agricultural sustainability in several planetary territories by restricting arable land and reducing the availability of water (Mickelbart et al., 2015). These obstacles, fastened with a lot of abiotic stresses like excessive temperature, salinity, drought, and cold, cause significant losses in crop yields and their subsequent socio-economic repercussions. For instance, abiotic stresses (e.g., salinity, drought, etc.) are accountable for regression in the production of major crops up to a range of 50-70% (Mittler, 2006). Further, over 800 million hectares of arable land worldwide are contaminated one or the other by excess saline (397 million hectares) or sodium (434 million hectares) contents (Munns, 2005). Mostly, crops are being affected by drought than any other stresses and, by dint of global climate change, are rising even more acutely in the world. Statistically, from 1970 to 2000, drought stress turned out to double globally (Isendahl and Schmidt, 2006). In the light of the predicted climate change scenarios, an extensive increase in the production of agriculture would be obligatory to act in accordance with the world's food needs for the ongoing growing population in the upcoming half-century (Myers et al., 2017;Shaar-Moshe et al., 2017).
Changes in different physiological and metabolic processes, however, are the most common instances under excessive salt and water deficit stress, depends on the period and extent of stress and consequently impede agricultural production (Rozema and Flowers, 2008;Rahnama et al., 2010;James et al., 2011;Apel and Hirt, 2004;Gill and Tuteja, 2010;Hasanuzzaman et al., 2014a). Numerous changes have occurred in plants subjected to salinity and drought stress, including membrane destruction, nutrient disparity, capability impairment to detoxify reactive oxygen species (ROS), decreased photosynthetic function, variations in enzymatic activity of antioxidants, and decreased aperture of stomata (Rahnama et al., 2010;Apel and Hirt, 2004;Gill and Tuteja, 2010;Munns and Tester, 2008;HanumanthaRao et al., 2016). In addition, excessive salt and water deficit stress results in uncontrolled production of ROS products, like superoxide (O 2 •− ), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), and radicals of hydroxyl (OH • ) (Apel and Hirt, 2004;Gill and Tuteja, 2010;Rohman et al., 2016a;Rohman et al., 2016b). The formation of stress-induced ROS can leash to oxidative damage in several components of the cell include proteins, lipids, and DNA, and also disrupting essential plant cellular functions (Gupta and Huang, 2014). Conversely, plants possess a unique core mechanism to adapt to ROS toxicity and are assumed to counter water deficit stress by reinforcing these core mechanisms of defense unless removed or declined in the cell (Hasanuzzaman et al., 2014a;Rohman et al., 2016a;Rohman et al., 2016b).
For growing plants, the approach of using plant growth regulators (PGRs) is showing the potential to tolerate different abiotic stresses nowadays (Ali et al., 2013;Ali et al., 2014). 5-aminolevulinic acid (ALA) is one of them, and it is an important PGR engaged in the modulation of growth, development, and numerous physiological responses (Akram and Ashraf, 2013). ALA is a key component and precursor of porphyrin-derivatives biosynthesis as well as is involved in essential physiological processes of plants, including respiratory, photosynthetic, and other metabolomics activities (Wang et al., 2003;Zhang et al., 2015). Amassing evidence suggests that ALA can combat abiotic stresses (salinity, drought) by inducting plant resistance (Ali et al., 2013;Korkmaz et al., 2010;. Nonetheless, underlying mechanisms remain to be established as to how ALA stimulates antioxidant systems to modulate plant stress resistance under the combination of different stresses. Moreover, plants must simultaneously cope with multifarious environmental stresses under both natural and agricultural systems and acclimatize with unrest environmental conditions. Under field conditions, salinity level increases with decreasing soil moisture. At the same time, salinity stress itself causes osmotic stress by raising the concentration of different ions in soil water. Therefore, plants encounter both saline and drought stress concurrently. However, the conventional method of working concentrates on short and severe single stresses of plant responses to abiotic stress in early vegetative phases, with plant endurance or retrieval levels as a magnitude for tolerance to plant stress. Plant acclimatization to multiple environmental stress combinations is more complex than to single stress and can generate certain physiological, molecular, and metabolic responses interacting and inhibiting each other (Suzuki et al., 2014;Prasch and Sonnewald, 2015). However, information on such responses under salinity and drought-mediated combined stress is still limited. C 4 type maize (Zea mays) plants belong to the family Poaceae. By nature, due to being a cross-pollinated crop, maize is supposed to be more adaptable owing to superior photosynthesis, compared to C 3 plants, although at the expense of reduced photorespiration (Kanai et al., 1999). Maize was therefore found to be experiencing the least oxidative stress. Nevertheless, a few numbers of recent studies have shown that maize is suffering extensively under abiotic stresses resultant oxidative damage, especially excessive salt or water deficit stress (Rohman et al., 2016a;Rohman et al., 2016b;Rohman et al., 2019;Anjum et al., 2017). However, the effect of combined stress in maize also inadequate. Therefore, we designed the study to assess the potential protection of ALA supplementation on maize seedlings in two different concentrations under specific salinity and drought and their combined stress. In the current work, we investigated the following key attributes: (i) performance on plant growth, (ii) status of photosynthetic pigment, (iii) accumulation of proline, (iv) Na + and K + homeostasis, (v) oxidative damage in relation to elevated levels of ROS and lipid peroxidation, and (vi) improved antioxidant protection.

Plant materials
Two high-yield maize varieties, namely BARI Hybrid Maize-7 (BHM-7) (yield: 10.5-11.2 t/ha) (BARI, 2020) and BARI Hybrid Maize-9 (BHM-9) (yield: 10.2-12.0 t/ha) (BARI, 2020), were used to study the attenuating effects of 5-AminoLevulinic Acid (ALA, Wako, Japan) under salinity, drought, and a combination of these stresses (drought and salinity). Seeds of both varieties were obtained from the Plant Breeding Division of Bangladesh Agriculture Research Institute (BARI), Joydebpur, Gazipur. After keeping the seeds in room temperature for 24 h, substantial healthy seeds were sown in a tray that was previously filled with a mixture of treated coarse sand and rock grinding and allowed to be kept in a greenhouse at 22°C for 7 days for germination. Afterward, the 7-day-old seedlings were subjected to grow in a controlled hydroponic condition (temperature: 22°C, relative humidity: 75-80%, light intensity: 370-555 µmol/m 2 /s) using Hoagland solution as a nutrient, which renewed in every 2 days.

Growth parameter measurement
The assessment of shoot height, root length, and volume were performed, using the technique defined by Rohman et al. (2019).

Determination of photosynthetic pigments and proline content
The chlorophyll (Chl) a, b, and Chl (a+b), and carotenoid contents were estimated spectrophotometrically in the penultimate leaves and calculated following the method proposed by Lichtenthaler (1987) and Arnon (1949), consecutively. Proline (Pro) content in the penultimate leaves of maize plants was quantified according to the protocol of Bates et al. (1973).
Measurement of Na + /K + To assess the sodium (Na + ) and potassium (K + ) contents, the sap was extracted from freshly harvested roots and shoots, using a tissue sap extractor (Horiba, Japan), and the contents were determined using the compact Na + ion (Horiba-731, Japan) and K + ion (Horiba-722, Japan) meters, respectively, following the methods stated in Rohman et al. (2019).

Assessment of the contents of superoxide (O 2
•− ) and hydrogen peroxide (H 2 O 2 ) In the penultimate leaves of maize, radical superoxide (O 2 •− ) and hydrogen peroxide (H 2 O 2 ) were determined following the procedures of Elstner and Heupel (1976) and Yu et al. (2003), correspondingly.

Histochemical ROS marker detection
To detect the localization of O 2 •− and H 2 O 2 in the penultimate leaves, the procedures of Chen et al. (2010) and Thordal-Christensen et al. (1997), respectively, were followed.

Determination of the level of protein
The concentration of protein in the leaf extracts was quantified applying bovine serum albumin (BSA) as a protein standard following the Bradford method (Bradford, 1976).

Native PAGE and staining of activity
To visualize the activities of several enzymes protein changes these possesses isoenzymes activity, and scavenge reactive oxygen species (ROS), also participate in the ascorbateglutathione cycle were studied in this study using PAGE (on a gel, 10% for SOD and 8% for others) maintaining nondenatured and non-reduced conditions at 4°C as described by Laemmli (1970). In each lane of the gel 50 µg of protein was applied. Procedures followed for gel running and staining are described below.
The method defined by Beauchamp and Friclorich (1971) was followed for detecting SOD activity. Prior to soaking the gel in 50000 µM Na 2 PO 4 buffer (pH 7.8) at room temperature for 20 min in shady condition, electrophoresis was done. The buffer includes nitroblue tetrazolium (NBT) (240 µM) and riboflavin (28 µM). Afterward, TEMED (28000 µM) was included in the buffer, and then the gel was kept under an artificial light source until the band was visualized.
The procedure of Woodbury et al. (1971) was adopted to determine CAT isozymes. After electrophoresis, gels were soaked into 0.01% H 2 O 2 for 5 min. After that, gels were rinsed with double distilled water. Finally, 1% FeCl 3 and 1% K 3 Fe(CN) 6 were used to stain the gels and to detect the isozymes.
To find out the APX activity, the process of Mittler and Zilinskas (1993) was tracked. First, for 30 min, the gel was pre-run adding 2000 µM L-ascorbic acid without loading sample. Followed by immersing the gel in 50000 µM Na 2 PO 4 buffer (pH 7) for 30 min; buffer contained 2000 µM L-ascorbic acid. Afterward, the gel was transferred to a solution composed of 50000 µM Na 2 PO 4 buffer (pH 7), containing L-ascorbic acid (4000 µM) and H 2 O 2 (2000 µM); the incubation period was 20 min. Subsequently, the gel was washed with 50000 µM Na 2 PO 4 buffer (pH 7) for 1 min. Lastly, the gel was submerged in 50000 µM Na 2 PO 4 buffer solution (pH 7.8), composed of TEMED (28000 µM) and NBT (2450 µM) for 15 min with gentle agitation.
POD isoenzyme activity was identified following the procedure of Fielding and Hall (1978). Potassium buffer (pH 7) (25000 µM) was used to soak the gel for 15 min. Then, the gel was infused into a newly prepared 25000 µM K-phosphate buffer (pH 7) solution until the POD activity band appeared, the buffer composed of guaiacol (18000 µM) and H 2 O 2 (25000 µM).
The technique described by Das and Bagchi (2010) was adopted for showing GPX activity. Washing of gel was performed with 2.5% Triton X-100 and double distilled water; in each case, the washing duration was 15 min. Next, for 1 h gel was soaked in 10000 µM K-PO 4 buffer (pH 7.2), 2000 µM o-dianisidine dihydrochloride was mixed in the solution. In the last step, the gel was moved to 10000 µM K-PO 4 buffer (pH 7.2), containing 100 µM H 2 O 2 . The gel was kept in that solution until brown GPX bands appeared in contrast to the pale-yellow background.

Statistical analysis
Data reported in the figures and tables were expressed as means with standard deviations (SDs) of three independent replications (n = 3) of each treatment; each was repeated two times. Data obtained in this work were analyzed out using the two-way analysis of variance (ANOVA) using the software Statistix (version 10.0). Different letters represent significant differences among the treatments following the least significant difference test to compare the treatment means at p ≤ 0.05.

Phenotypic appearance
Exposure to salt, drought, and combined stress (salt+drought) condition severely damaged maize seedlings. By phenotype, resultant rolling of lower leaves, leaf tips burning, and, in certain cases, of the entire leaves, leaves drooping and yellowing of the whole plant. The most devastating result was found in the combined stress condition where maize seedling almost failed to survive. Supplementation of 5-aminolevulinic acid (ALA) at low concentration (e.g., 20 µM) notably overturned the stress-induced obliteration and amended the seedling's phenotypic appearance. More damaged seedlings were observed when seedlings were supplemented with a higher concentration of ALA (80 µM) compared to the low concentration ( Fig. 1). However, the stress-induced damage was more visible in BARI Hybrid Maize-7 (BHM-7) than BARI Hybrid Maize-9 (BHM-9) variety.

Plant growth
The growth of plants considering shoots height, root length, and root volume was decreased due to stresses. Highly reduced growth was found under the combined stress condition; where, shoot height, root length, and root volume, were diminished by 237.0, 23.0 and 296.7% in BHM-7; whereas, 152.5, 41.0 and 127.5% in BHM-9 compared to control seedlings (Tab. 1). Nevertheless, supplementation of 20 µM exogenous ALA to stresses-treated seedlings distinctly repaired the growth of plants. The restoration percentage of shoot height of BHM-7 was 11.1, 7.2 and 104.8%; root volume was 31.1, 7.0 and 151.7%; while, for BHM-9 shoot height refurbished by 48.2, 19.8 and 46.7%; root volume by 22.0, 48.8 and 23.5% in comparison with their respective stresses (Tab. 1). On the other hand, applying 80 µM ALA to the salt, drought and combined stress treatment reduced the growth of plants once again, which points out that a higher concentration of ALA acts as a suppressor to the plant growth. However, plant growth was highly affected in the BHM-7 variety than BHM-9 variety (Fig. 1). An exception was found in the case of root volume in BHM-7 under sole drought stress, where volume was higher than BHM-9; this because during estimation we observed more adventitious roots in drought-treated BHM-7 seedlings.

Photosynthetic pigments
Salt exposure of BHM-7 and BHM-9 maize seedlings resulted in decreased chlorophyll (chl) a (by 35.5 and 29.3%, accordingly), chl b (by 71.3 and 55.1%, accordingly) and chl (a+b) (by 50.3 and 39.6%, accordingly), compared to control seedlings. Further, drought stress reduced the contents of chl a (by 9.2 and 195.6%, individually), chl b (by 104.5 and 184.3%, individually), and chl (a+b) (by 39.9 and 190.5%, individually) in BHM-7 and BHM-9 seedlings, correspondingly, compared to that of control plants. However, in contrast to that of the control condition, maize seedlings treated with combined stress showed declination in the levels of chl a (by 54.7 and 87.2%, separately), chl b (by 144.0 and 130.5%, separately), and chl (a+b) (by 79.7 and 104.3%, separately) in BHM-7 and BHM-9 varieties, respectively (Tab. 2). Exogenous application of ALA to the salt, drought and combined stress-treated maize seedlings amended chl content compared to the seedlings treated with only salt or drought and in combined. Although, applying the ALA in higher concentration improved chl content in some treatment but decreased in others.
Carotenoid and proline (Pro) content Under saline stress, BHM-7 and BHM-9 maize seedlings showed reduced carotenoid content (by 109.2 and 35.8%, accordingly), compared to control seedlings. Additionally, drought stress decreased the carotenoid contents (by 40.0 and 31.0%, individually) in BHM-7 and BHM-9 seedlings, correspondingly, compared to that of control plants. Nevertheless, in distinction to that of the control condition, combined stress treated maize seedlings exhibited a reduction in the level of carotenoid by (71.0 and 41.0%, separately) in BHM-7 and BHM-9 varieties, respectively (Tab. 3). Exogenous ALA application to the salt, drought, and combined stress-treated maize seedlings revised carotenoid content compared to sole salt or drought and in combined seedlings. Though, ALA supplementation in 80 µM concentration amended carotenoid content in some cases but diminished in other treatments. All the stresses markedly escalated in endogenous proline (Pro) content in maize seedlings of both varieties; however, the percent increase was higher in BHM-9 compared to BHM-7. In BHM-7 Pro content increased by 23.2, 18.7, and 20.5% under salt, water deficit, and combined stress, correspondingly, compared to that of control; in the same sequence of stress, BHM-9 showed 81.8, 625.0, and 359.0% increase (Tab. 3). Importantly, supplementation of ALA at 20 µM concentration decreased the endogenous Pro content under salt stress but increased in water deficit and combined stress than the control seedlings. Nevertheless, whenever ALA was applied at 80 µM concentration endogenous Pro content slightly increased in salt, whereas a significant increase occurred under water deficit and combined stress than the control seedlings.
Na + and K + homeostasis A markedly higher Na + uptake and Na + /K + ratio was observed in the shoot and root of both varieties treated with saline, drought, and combined stress compared to that of control (Figs. 2A-2F). Conversely, K + reduced in both varieties. Exogenous ALA supplementation at 20 µM concentration to salt caused a significant decrease in Na + uptake in the shoot of maize seedlings by 853.9 and 836.5%, respectively (Fig. 2B). Accordingly, it decreased Na + in the root by 52.8 and 53.0% in BHM-7 and BHM-9, respectively ( Fig. 2A). Conversely, it increased K + uptake in the shoot by 51.8 and 41.8% and in the root by 35.8 and 69.7% in BHM-7 and BHM-9, respectively. Similar results were found for drought and combined stress (Figs. 2C and 2D). However, ALA at 80 µM was not so effective in single stress (Figs. 2A-2F). Importantly, in combined stress, it significantly reduced the Na + and Na + /K + ratio as well as increased K + in both varieties.
Reactive oxygen species (ROS) generation, lipid peroxidation (MDA) and lipoxygenase (LOX) activity The salt, drought, and combined stress triggered an excessive generation of reactive oxygen species (ROS) in the maize seedlings compared to control, resultant boosted lipid  ) and hydrogen peroxide (H 2 O 2 ) through dark blue spots (Fig. 3A) and brown spots (Fig. 3B), accordingly, in the stress-treated seedlings; spots were more evident in BHM-7 than BHM-9. Supplementation of ALA, especially 20 µM, significantly lessened the spots of O 2 •− and H 2 O 2 in stress-treated seedlings.

Activities of antioxidant enzymes
As compared to control, superoxide dismutase (SOD) activity increased in BHM-7 by 50.5, 34.3 and 67.1% in salinity, drought and combined stress, respectively; likewise, in BHM-9 by 47.2, 27.4 and 35.5% in seedlings exposed to salt, drought, and combined stress, respectively (Fig. 4A). Interestingly, exogenously 20 µM ALA application decreased SOD activity significantly in both varieties compared to salinity, but in drought, the activity decreased only in BHM-7. In combined stress, the activity decreased in both varieties, being significantly lower in BHM-9. In contrast, ALA at 80 µM concentration maintained higher activities in all stresses than those at 20 µM concentration (Fig. 4A). Importantly, at higher ALA concentration, BHM-9 showed significantly higher SOD activity than BHM-7 in both drought and combined stress. The changes in the activity levels were also supported by in-gel SOD activity (Fig. 5A).
The maize seedlings, exposed to different stresses had declined catalase (CAT) activity compared to that of control (Fig. 4B). In contrast, 20 µM ALA supplementation significantly stimulated CAT activity in salt-treated BHM-7 FIGURE 3. Effects of exogenous ALA on reactive oxygen species buildups and lipoxygenase activity in leaves of maize seedlings exposed to salt, drought, and combined stress for 7 days. and BHM-9 maize seedlings (by 112.3 and 140.9%, respectively), in drought (by 136.0 and 284.4%, respectively), and in combined stress (by 351.1 and 116.2%, respectively), while comparing with the seedlings treated with respective stress. On the contrary, exogenous supplementation of ALA at the rate of 80 µM in stress conditions decreased the CAT activity significantly in comparison with 20 µM ALA treated maize seedlings (Fig. 4B). The in-gel activity of CAT showed a similar banding pattern of the CAT isozymes (CAT1 and CAT2) (Fig. 5B). Though CAT1 was not so clear, CAT2 intensity was greatly stimulated in leaves exposed to 20 µM ALA treatment.
Four bands of ascorbate peroxidase (APX) isoforms in the leaves of maize seedlings can be detected (Fig. 5C). In all isoforms band of stresses and 80 µM ALA treatment showed pronounced isozyme activity. The maize seedlings revealed to salt, drought, and combined-stress had escalated APX activity by 35.1, 21.1 and 54.4% in BHM-7, and, by 38.2, 29.1 and 135.3% in BHM-9, respectively, compared with control (Fig. 4C). Nevertheless, exogenous application of ALA at 20 µM decreased APX activity under salt stress in both varieties but again increased at 80 µM treatment, in contrast to solely salt-treated maize seedlings. However, in drought and combined stress conditions when ALA was applied at 20 µM concentration APX activity decreased in the BHM-7 variety but increased in the BHM-9 variety. On the other hand, vice versa result was found at 80 µM concentration. Isozymes (APX1, APX2, APX3, and APX4) of APX gave similar changes in the activity by changing band intensity in the gel (Fig. 5C).
Imposition of salt, drought and combined stress radically enhanced the peroxidase (POD) activity by 18.2, 39.0 and 61.0% in BHM-7, while, in BHM-9 it rose by 78.2, 65.5 and 101.8%, respectively, compared to control (Fig. 4D). Exogenous ALA supplementation at the rate of 20 µM in stressed seedlings reduced the POD activities drastically, compared to that of stress condition; whereas, 80 µM exogenous ALA application increased the POD activity significantly even higher than the respective stress treatment (Fig. 4D). In SDS-PAGE, changes in expression of POD isozymes (POD1 and POD2) were found in different treatments (Fig. 5D). However, the appearance of bands was diversified in BHM-7 and BHM-9 variety; it may be because of differences in varietal response.
In contrast to control seedlings, glutathione peroxidase (GPX) activity augmented significantly in BHM-7 variety under salt, drought and combined stress-treated seedlings by 74.7, 71.5 and 181.4%, respectively. Similarly, in BHM-9 variety by 47.8% in salinity and by 53.8% in combined stress. On the contrary, adding ALA at 20 µM concentration lessened the GPX activity in all treatments, but slightly boosted at 80 µM ALA concentration treatment in comparison to that of stresses alone (Fig. 4E). A similar banding pattern of GPX isozymes (GPX1, GPX2 and GPX3) was found in gel electrophoresis (Fig. 5E). In addition to this, in combined stress treatments, the GPX activity band was found to be highly induced in salinity and combined stress, particularly with 80 µM ALA (Fig. 5E).
Glutathione S-transferase (GST) activity in maize seedlings subjected to stress conditions was not uniform for BHM-7 and BHM-9 variety. On the one hand, in BHM-7 salt stress resulted in decreased GST activity, then, increased in drought and combined stress; then again, the augmented activity of GST in BHM-9 was found only in combined stress condition (Fig. 4F). Outstandingly, compared with the respective stresses, GST activity increased in BHM-7 by 111.0% in drought, by 84.4% in combined stress; whereas, in BHM-9 by 740.1% under salinity, by 55.3% under drought stress, in exogenous 20 µM ALA supplementation treatment; nevertheless, drastically decreased in 80 µM ALA treatment except for salt stress in BHM-7 (Fig. 4F).

Discussion
Plants exposed to salinity, drought, and combined stress condition causes osmotic stress, and inhibits the growth, by instigating higher Na + accumulation and interrupting ion homeostasis (Tuncturk et al., 2008;Munns, 2011). In the current research, stresses ensuing growth inhibition of maize seedlings in respect of shoot height, and root length and volume were noted, which were found to be renovated with a low concentration of ALA (20 µM) supplementation ( Fig. 1; Tab. 1). The improvement of growth inhibition convincingly supported by our findings of amended ion homeostasis and suppressing water deficiency caused by osmotic stress, which is consistent with previous findings (Nunkaew et al., 2014). Plant growth improvement by low concentration ALA treatment was also elucidated by Hotta et al. (1997), besides, in our study herbicidal response was found at 80 µM, and it was validated by the phenotypic outcomes of the current study.
Abiotic stresses affect the photosynthesis negatively by degrading the photosynthetic pigments, causing leakage in Chl molecules, and also reducing Chl contents (Kumar et al., 2017;Mehta et al., 2010). Certainly, in this study, maize seedlings subjected to stresses showed notable losses of Chls (Tab. 2) and carotenoid (Tab. 3). These results revealed that under stress conditions due to interrupted photosynthetic activity photosynthetic pigments supply was also disturbed, and consequently decreased the growth and caused chlorosis (Fig. 1). Nevertheless, after the application of ALA, the content of Chls increased, even more than the control level. As we know, Chl is the successor of the ALA precursor (Schlicke et al., 2014), and in this study, Chl biosynthesis was promoted by the supplementation of ALA. Ultimately, results showed that both 20 µM and 80 µM ALA supplementations improved the level of Chls of maize seedlings exposed to salt, drought, and combined stress (Tab. 2). Moreover, abiotic stresses promote chlorophyllase enzyme activity and/or boost ROS overproduction, and causes disruption of pigment-protein complex and lessen the photosynthetic pigments (Hasanuzzaman et al., 2014b;Saha et al., 2010), and in this study, supplementation of ALA restored and/or increases Chl contents of the leaf, it may by decreasing the activity of chlorophyllase enzyme.
Soluble proteins and Pro are vital osmotic protective elements in plants, they help to reduce osmotic potential under abiotic stresses (Arndt et al., 2001). Stresses affected plants uptake different inorganic ions, produce and pile up numerous osmoprotective substances and trivial organic molecules (Qados, 2011). Pro is crucial to fight against abiotic stresses, as it possesses osmoprotective and antioxidative potential (Nahar et al., 2016). Its concentration in cytosol increases with the exposure to abiotic stresses, as a result, water potential in cell decreases, and absorption of water increases (Kasuga et al., 1999). Since, salt and drought stress causes physiological water shortage by means of interrupted water absorption and exosmosis, which results from osmotic stress and ion toxicity (Munns, 2011); in our study, maize seedlings exposed to stress showed elevated Pro content (Tab. 3), signifying stress-induced osmotic stress and malicious water content. This outcome is sturdily reinforced by Hasanuzzaman et al. (2014b). However, exogenous ALA application at 20 µM reduced Pro deposition in the cell might be due to relief to some extent from all types of stresses as phenotypic appearances of maize seedlings exposed to low ALA treatment had better phenotype than that in stresses without ALA ( Fig. 1; Tab. 3). On the contrary, a higher dose of ALA showed the opposite result and proved its detrimental effect on plant growth. However, consistent result regarding the role of ALA on soluble proteins and Pro accumulation under salt and drought stress was also testified by Kosar et al. (2015) and Akram et al. (2012). Na + -induced K + efflux is the instant and prime reaction from plants subjected to an elevated level of salts (Anschuetz et al., 2014;Bose et al., 2014). Salt and drought-induced stress disrupt the ratio of Na + /K + (Simaei et al., 2012;Ali and Rab, 2017), and homeostasis of ions (Tuncturk et al., 2008). In the study, salt, drought, and their combination-induced stress raised Na + content and declined K + content in maize seedlings' shoots and roots, which could result from the engross of greater levels of Na + into the plant (Fig. 2). Higher accumulation of Na + also causes a higher ratio of Na + /K + , which interferes with ion homeostasis by lowering the content of Mg, Mn, and Zn. The Na + influx and K + leakage possibly be also for higher ROS production, as our result showed. A similar disruption of ion homeostasis was demonstrated in previous studies under elevated salt and deficit water stress conditions (Ali and Rab, 2017;Demidchick and Maathuis, 2007;Wu and Wang, 2012). It was noted, nevertheless, that exogenous ALA at low concentrations decreased the Na + concentration in the cotton plant roots, and it was assumed that in growth media the manifestation of ALA could lead to a decrease in Na + uptake and could overcome water shortage due to osmotic stress induced by high Na + concentration nearby the roots (Watanabe et al., 2000). Our results are also steady with the findings of Naeem et al. (2010); ALA applied to oilseed rape seedlings, grown under high saline stress drastically decreased Na + and K + accumulation, resulting in reduced Na + /K + ratio in both roots and leaves than the control plants. Moreover, it has also been stated that elevated Na + buildup in the leaves in contrast to roots indicates that ALA does not stimulate Na + transportation from the roots to the shoots, but somewhat may withhold Na + ions uptake to the roots from the growth media. Youssef and Awad (2008) reported that decreased Na + accumulation in the leaves and lessened K + uptake by the roots, which resulted in a concurrent reduction in the K + /Na + ratio. However, higher Na + and Na + /K + ratio in single stresses at 80 µM ALA than 20 µM is not clear. One of the causes might be that higher ALA in single stress can encourage Na + uptake, and transportation of higher Na + can be arrested in xylem, another salt tolerance mechanism of the plant (James et al., 2006), because those values in the leaf were comparatively lower at that concentration of ALA ( Supplementary Fig. 1). In the present study, exogenous application of ALA at 20 µM concentration amended ion homeostasis by reducing Na + uptake and ROS generation, as verified by our results; or may be by relaxing the effects of salt and drought on maize seedlings (Fig. 2), as depicted by Ali and Rab (2017), and Watanabe et al. (2000). In contrast, external supplementation of ALA at higher concentration showed damaging effects on plants (Hara et al., 2011), consistent result with this statement has been found in this study in the case of exogenous ALA application at 80 µM treatment (Fig. 2). Nevertheless, the role of ALA on Na + /K + ratio and ion homeostasis on different plants exposed to abiotic stresses was reported previously in several studies (Ali et al., 2014;Liu et al., 2014;Akram and Ashraf, 2011); which are consistent with our present results.
Abiotic stresses alter plants with an adverse effect on the gas exchange, resultant in low CO 2 integration for photosynthesis and consequently a substantial drop in electron transport ensuing in increased ROS production (Gill and Tuteja, 2010;Møller et al., 2007). In both cases, the generation of ROS is enhanced, which is extremely cytotoxic to cell organelles such as protein, DNA, lipid, and pigment (Gill and Tuteja, 2010). In this analysis, substantially elevated ROS (O 2 •− , H 2 O 2 ) was observed in salt, drought, and combined stressed seedlings than the control treatment (Figs. 3A-3D), indicated oxidative eruptions in maize leaf tissues. Elevated ROS causes lipid peroxidation, a latent biomarker of oxidative damage to the membranes, leading to electrolytes leakage, loss of membrane permeability, and degradation of membrane proteins and ion channels (Munns and Tester, 2008;Turan and Tripathy, 2013;Nedjimi, 2014). In the current study, elevated ROS caused higher lipid peroxidation product (MDA) together with its associated enzyme, LOX (Fig. 3E,  3F). Thus, elevated ROS and MDA in maize seedlings can impair the root and shoot tissue. Our previous study also documented oxidative damage in maize resulted from salt stress (Rohman et al., 2019). However, a number of researchers documented significantly higher rates of O 2 •− , H 2 O 2 , and MDA than controls in mustard Ahmad et al., 2012), cucumber (Li et al., 2011), and maize (Rohman et al., 2018), under salt and drought stress. Exogenous ALA was also reported to mitigate ionic imbalance, ROS bursts, and MDA content under salinity in beet seedlings (Liu et al., 2014), and under drought in cucumber seedlings (Li et al., 2011). Under abiotic stress, including salinity, ALA mediated changes in some of the main physiological attributes in plants and accumulates sugars and other corresponding solutes that assist as an osmoprotectant and, in certain cases, stabilized biomolecules under stress conditions   (Gill and Tuteja, 2010). However, the higher concentration of ALA itself can also increase the density of growing media to create osmotic stress.
In the present study, the application of ALA at a concentration of 20 µM demoted salinity, drought, and combined stress-mediated elevated concentrations of O 2 •− , H 2 O 2 , and MDA and activity of LOX in all stress treatments (Fig. 3). In all stress's MDA production was clearly linked to ROS generation and LOX activity. This is in line with other salinity and drought-imposed studies Ahmad et al., 2012;Zhen et al., 2012). Link amid these can be clarified by taking the antioxidative activities in mind. In addition, ALA mediated LOX activity inhibition is expected to diminish the generation of MDA in the maize leaf. On the contrary, ALA at higher concentration (80 µM) acted as the stress treatments alone, as a result, it increases ROS production (O 2 •− and H 2 O 2 ), prompted MDA content and enhanced the LOX activity. Our findings are in harmony with the study of Liu et al. (2011). Definitively, in this study, low ALA treatment imposed to salt, drought, and combined stress, protected the plants by minimizing the ROS and MDA production, as well as, by reducing the LOX activity, which strongly supports our other findings, like, plant growth and lower Na + /K + ratio; conversely, supports the herbicidal effect of ALA at higher concentration.
Among the antioxidant enzymes, SOD is thought to be a first-line enzymatic defense in regulating ROS, which alters O 2 •− to H 2 O 2 (Gill et al., 2015), and then CAT detoxifies H 2 O 2 readily to H 2 O and O 2 (Sanchez-Casas and Klesseg, 1994). The present work exhibited a substantial rise in SOD activity and a decline in CAT activity under conditions of salt, drought, and combined stress (Fig. 4A, 4B), these are consistent with earlier reports (Mishra et al., 2013;Wutipraditkul et al., 2015). This improved SOD activity may be attributed to increased production of O 2 •− and H 2 O 2 and reduced CAT activity as a result of greater production of H 2 O 2 , which was caused by oxidative stress, this result is approvingly supported by former research works (Rohman et al., 2016a;Rohman et al., 2016b;Hasanuzzaman et al., 2014b;Mishra et al., 2013;Nahar et al., 2015). Most interestingly, the exogenous application of ALA showed two different results in two different concentrations for SOD and CAT activity. Where, 20 µM ALA decreases SOD activity, but, increases CAT activity significantly; on the other hand, the supplication of ALA at 80 µM concentration enhanced SOD activity, while, reduces CAT activity under stresses. From this, it can be suggested that CAT is the most active antioxidant, which respond significantly in low concentration ALA treatment during this experiment, which greatly supports the morphological appearance of the stressed plant and minimized production of O 2 •− and H 2 O 2 . A parallel result was not reported yet. However, several results were found previously concluded that ALA supplementation increased both SOD and CAT activities and scavenged ROS simultaneously Zhang et al., 2013;Niu et al., 2017). As CATs are tetrameric heme-containing enzymes so exogenous ALA application might have some direct relation with CAT activity but the underlying process is yet to find. Further, in gel electrophoresis the expression of SODs and CATs were detected (Figs. 5A, 5B), which strengthened the present findings.
Ascorbate peroxidase is one of the essential enzymes of the AsA-GSH cycle and acts against oxidative stress along with AsA and GSH by reducing the generation of ROS and recycling of AsA and GSH (Mishra et al., 2013). Ascorbate peroxidase converts H 2 O 2 to produce H 2 O by the oxidation of AsA to DHA. In the current study, the APX activity increased with salt, drought, and combined stress (Figs. 4C and 5C). The stimulation of this behavior of the antioxidant enzyme suggests its association with oxidative stress tolerance (Mishra et al., 2013). The improved activity of APX may have been attributed to the higher content of H 2 O 2 . This observation is consistent with earlier studies (Hasanuzzaman et al., 2014b;Mishra et al., 2013;Ozfidan-Konakci et al., 2015). However, the exogenous ALA application showed a confusing result. In both varieties, ALA at 20 µM decreased APX activity under salt stress but boosted at 80 µM treatment contrasted to only salt-treated maize seedlings. Conversely, in drought and combined stress conditions when ALA was applied at 20 µM concentration APX activity decreased in BHM-7 variety but increased in BHM-9 variety. On the other hand, vice versa result was found at 80 µM concentration. Maybe the reason behind this, the activity of different antioxidants varied in different varieties.
A heme-containing enzyme is POD, which is essential in stressed conditions for balancing of reactive intermediate products of O 2 and peroxide radicals (Vangronsveld and Clijsters, 1994). In this analysis, salinity, drought, and combined stress in both varieties significantly induced POD activity compared to control activity (Figs. 4D and 5D). POD, therefore, is crucial in the metabolism of ROS and thus conferred tolerance under all the stresses on maize seedlings. In our previous research, the induced behavior of POD under salinity and drought was also recorded in maize (Rohman et al., 2019), mustard Ahmad et al., 2012), and cucumber (Li et al., 2011;Zhen et al., 2012). In this study, in all stresses while ALA was applied at 20 µM concentration inhibited the activity, but salinity; on the other hand, at higher concentration ALA enhanced POD activity significantly, but in the drought condition of BHM-7. Although increased activity was documented in several studies due to ALA application in salinity and drought treated plants (Ahmad et al., 2012;Li et al., 2011); however, ALA mediated this form of POD activity in combined stressed maize or other plants has not been documented until now.
The GPX and GST enzymes function collectively to defend plants by creating comparatively less noxious and conjugates, which are water-soluble under oxidative stress through catalyzing the bonding of various xenobiotics and their electrophilic metabolites (Gill and Tuteja, 2010). In this study, the stress-persuaded oxidative stress redoubled GPX activity, nonetheless, declined GST activity (Figs. 4E, 4F and 5E). On the one hand, this increased GPX activity may be because of over-production of H 2 O 2 and its' inadequate detoxification; on the other hand, reduced GST activity is due to increased GPX activity, because, as we know, GPX uses GSH for its activity, and GSTs works by GSH conjugation (Cummins et al., 2011). This result is partially consistent with Rohman et al. (2016b), and Hasanuzzaman et al. (2014b), who described the escalated activity of GPX and GST under saline-induced oxidative stress situations. However, exogenous ALA application at 20 µM in the stress-treated maize seedlings decreased GPX activity, but increased GST activity (Figs. 4E, 4F and 5E); increased GST might play a role in detoxifying H 2 O 2 and in apoptosis to protect the plant. In addition, GST assisted in herbicide detoxification in high concentrations of ALA and this is why GPX activity increased. Dixon et al. (2010), and Mostofa et al. (2019) strongly support this result.
To sum up, not only we found that low concentration of ALA has significant optimistic role in plants but also its detrimental effect at higher concentration. An unswerving result was found by Liu et al. (2011), on mustard plant. They said that ALA treatment at higher concentrations killed the plants. Their finding importantly assists the result of the present study, where 80 µM ALA with salt and drought stress hampered the plant growth notably.

Conclusions
It may be deduced from this study that, salt, drought, and combined stress disrupt the antioxidant defense system of plants by increasing ROS production. Maize seedlings received the highest amount of damage under the combined stress condition; phenotypic appearance, morphological, physiological, and biochemical data confirmed that. ALA application exogenously at 20 µM concentration to stresstreated maize seedlings increased the detoxification of overproduced ROS by strengthening antioxidant protection specifically CAT function, excluding salt and drought stressexerted oxidative stress; additionally, GST might have an imperative function in herbicidal detoxification generated by ALA application. However, under stress conditions, a lower dose of ALA showed a depressing response to most of the activity of antioxidant enzymes, except CAT and POD (in salt). Contrarily, a higher concentration of ALA improved the enzymatic activity of antioxidants (SOD, POD, APX, and GPX) but failed to ROS scavenging, which proved that exogenous ALA had a positive effect when applying at low concentration but demonstrate detrimental result when applying at higher concentration. Moreover, the gel activity of SOD, POD, CAT, APX, and GPX inveterate these findings. Importantly, high concentration application of ALA under combined stress has played a vital role in increasing antioxidant activity by destroying plant roots and preventing a greater accumulation of ALA in plants. So, a higher concentration of ALA application in the combined stress condition acted as a low concentrated ALA.