Thiamine and Indole-3-Acetic Acid Induced Modulations in Physiological and Biochemical Characteristics of Maize (Zea mays L.) under Arsenic Stress

Abstract

Arsenic (As) accumulation in plant tissues is an emerging threat to crop productivity and global food security. As-toxicity in soil is increasing at alarming rates through mining, pesticide applications and industrial revolution. Therefore, a novel study was conducted to disclose the role of vitamin B1 (thiamine) and Indole-3-acetic acid (IAA) in maize (Zea mays L.) against different As stress levels, i.e., 0, 50 and 100 mg/kg sodium arsenite (NaAsO₂). Seeds of two contrasting maize varieties Akbar and Pearl were primed with different treatments, i.e., control (non-primed seeds), thiamine (250 ppm), IAA (30 µM) and a mixture of thiamine (250 ppm) + IAA (30 µM). Of both As stress levels (50, 100 mg/kg), a higher As stress level (100 mg/kg) imparts maximum negative impacts on maize growth by decreasing shoot and root nutrient ions—potassium (K), calcium (Ca), phosphorus (P), total phenolics, total soluble proteins—as compared to the control, while increases in catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), malondialdehyde MDA, hydrogen peroxide (H₂O₂), proline, total soluble sugars, free amino acids and ascorbic acid were recorded under As-stress as compared to control. The varietal differences showed that Pearl exhibited tolerance against As-stress as compared to Akbar. However, thiamine and IAA treated seeds of maize manifested remarkable enhancement in plant growth attributes with better chlorophyll, plant water status, enzymatic and non-enzymatic antioxidants activities under imposed As-stress. The growth and biomass significantly increased with priming treatments of thiamine and IAA under both As-stress levels of both varieties that suggests the role of these in As-stress tolerance. Overall, the performance order in improving growth under As-stress of thiamine and IAA treatments was thiamine + IAA > thiamine > IAA observed in both maize varieties.

1. Introduction

Arsenic (As) is considered as the most toxic environmental pollutant that causes severe damages to crops when it enters into the soil environment through sewage and industrial waste irrigations (Otero et al., 2016 [1], Kayode et al., 2021 [2]). Petrochemical industries and oil refineries are the major sources that contaminate soils with As (González et al., 2021 [3]). Coal combustion, metallurgical, and artistic glass industries are the other sources of massive As emission into the natural environment (Zevenhoven et al., 2007 [4]; Formenton et al., 2021 [5]).

Arsenic occurs in As (III) and As (V) as the most toxic forms in the natural environment. Both these forms are dominant and inter convertible, depending upon chemical and biological processes. As (III) is a soluble form of arsenic (Itaya et al., 2022 [6]) and dominates in anaerobic environments, while As (V) is mostly found in a solid state under aerobic conditions (Smedley et al., 2002 [7]; Sharma and Sohn 2009 [8]; Cai et al., 2020 [9]). As (V) holds a high affinity with P-transporters, enters into the plant system through a structural analogy with phosphate and competes with Phosphorus (P) by diminishing ATP production that results in stunted growth (Sharma et al., 2020 [10]). The soluble form of As (III) enables it to associate with fertile soils and increases its bioavailability for plants via contaminated soils. As (III) is a hundred times more toxic than As (V) and binds with protein sulphydryl groups and degraded membranes and even causes cell death (Bhattacharya et al., 2021 [11]). The extensive As transport through these channels competes with nutrient ions and disturbs essential nutrients’ ion uptake that causes nutrient deficiency in plants (Das et al., 2020 [12]).

Arsenic toxicity inhibits the physio-biochemical, cellular and molecular functions of plants (Ulhassan et al., 2022 [13]) and is a death dealing agent even at a very low quantity of about 7 mg L⁻¹ (De Oliveira et al., 2018 [14]). Extensive studies prove that As-contamination negatively affects the seed germination and seedling growth (Mridha et al., 2021 [15]; Nouri et al., 2021 [16]; Kumar et al., 2022 [17]). Excessive levels of As disturb the plant water relation and gas exchange attributes due to oxidative damages in plants (Arikan et al., 2022 [18], Shah et al., 2022 [19]). It alters the root metabolism and triggers the overproduction of reactive oxygen species (ROS) (Choudhary et al., 2020 [20]). These over-accumulated ROS are the prominent cause of negative impacts on plant systems (Berni et al., 2019 [21]). The instigated ROS levels impose oxidative stress that damages the cell structure, i.e., nucleic acids, proteins and lipids (Shahid et al., 2021 [22]). The antioxidant system is also dependent on ROS concentration and works with coordinate manners (Suhel et al., 2022 [23]; Fatima et al., 2021 [24]). Therefore, high levels of As results in increasing levels of ROS that consequently damage the entire system of plants. Additionally, over expression of ROS also increases the levels of antioxidant enzymes (CAT, POS, SOD, APX) and membrane lipid peroxidation MDA and H₂O₂ in plants (Suhel et al., 2022 [23]) that damage the cell structures.

Meanwhile, the accumulation of secondary metabolites (flavonoids, phenolics, ascorbic acid) is the defensive strategy to lower down oxidative damages (Bhat et al., 2022 [25], Shamshir et al., 2022 [26]). The amino acids tend to increase under As induced stress [27,28]. The ascorbic acid and phenolics are also secondary metabolites that have an important role in ROS scavenging under oxidative stress (Mishra et al., 2012 [29]). In addition, secondary metabolite production is considered as a stress coping strategy during the growing stages of plants (Isah 2019 [30]), while other compounds such as carotenoids and chlorophyll pigments decrease under higher As regimes while increasing under low As concentrations. The decreased levels of these pigments as a result of As toxicity decrease not only the growth of plants but also affect the crop productivity.

The maize (Zea mays L.) crop is the third most grown crop all over the world for different purposes and fulfills the needs of both animals and human (Bairwa et al. [31]). It is cultivated as feed and fodder crop all over the world, and the demand for maize crop is increasing rapidly day by day. However, maize production is facing serious challenges of changing environments and heavy metal contamination of soil (Aftab et al., 2020 [32]; Irfan et al., 2021 [33]). Arsenic is the top prior hazardous metalloid that decreases the growth and productivity of maize at alarming rates (Kaya et al., 2020 [34]; Khan et al., 2022 [35]). Pakistan is at the fourth position in maize cultivation that is facing the definite reduction in yield due to such heavy metals and metalloids’ contamination. Keeping in view the ecofriendly and economically suitable methods, there is a firm need to address As toxicity related hazards.

To control As stress has become challenge for a better and sustainable agri-ecosystem that associates with crop productivity and growth (Khalid et al., 2020 [36], Shahid et al., 2021 [22]). However, to overcome the challenges of reduced crop yield, several techniques have been adapted to cope with heavy metal stressed conditions (Pavlikova et al., 2008 [37]). The most recent strategy of seed priming was recognized as effective against biotic and abiotic stresses. Seed priming is a cheap and easily approachable technology adapted by the previous researchers under stressed environmental cues (Jisha et al., 2013 [38]). In the context of previous studies, seed primed crops with various substances proved to be effective against abiotic stress (Hussain et al., 2019 [39]).

The thiamine and IAA seed priming approaches may be a better and easy way to mitigate the detrimental effects of crops growing in As hosted soils’ environment. Previous studies reported that the continuous synthesis of thiamine or its enhanced endogenous levels was supposed to improve the plants’ tolerance against harsh stressed conditions (Abidin et al. 2016 [40]). Thiamine is associated with abiotic stress tolerance (Jabeen et al., 2021 [41]; Kaya et al., 2015 [42]; Ghaffar et al., 2019 [43]) and helpful in the synthesis of nucleic acids, carbohydrates, adenosine triphosphate and nicotinamide adenine dinucleotide phosphate (Nosaka 2006) [44]. Moreover, the alternative role of thiamine was recognized as a plant defense activator (Ahn et al., 2005 [45]). Under stressed conditions, thiamine proved to increase germination percentage in maize (Kaya et al., 2015 [42]). Being a vitamin of prime importance, it is introduced in medicinal plants for better nutrition and the treatment of thiamine deficiency related diseases (Sunarić et al., 2020 [46]).

On other hand, the auxins are considered as the organic phyto-hormones that coordinate with developmental processes in plants (Strydhorst et al., 2018 [47]). Indole-3-acetic acid (IAA) is a plant hormone of the auxin class that increases the photosynthesis in plants and accelerates the source-sink relation (Khan and Mazid 2018 [48]). IAA induces positive responses against heavy metals (Cd, Pb) and reduces lipid peroxidation MDA with an increase in chlorophyll pigments (Peat et al., 2012 [49]; Ji et al., 2015 [50]) that resultantly increases maize plants’ biomass (Hadi et al., 2010 [51]). Being the main individual of the auxin class and plant growth regulator, IAA has functions in a harmonized manner with other growth regulators (Amoanimaa-Dede et al., 2022 [52]). In addition, it improves the major developmental processes in plants and is involved in the induction of tolerance against various abiotic stresses (Chen et al., 2020 [53]). Moreover, it heals the oxidative injuries against As-stress and imparts better growth (Alamri et al., 2021 [54]).

Therefore, thiamine and IAA as priming agents on maize seeds under As-stress as a combined application were tested. To unlock the hidden role of thiamine and IAA as priming agents under As-stress, the experiment was designed to study two genetically different maize varieties to elucidate the biochemical mechanisms involved to ameliorate the negative role of As in the specific context of growth, oxidative stress, gas exchange, lipid peroxidation and ionic assimilation improvements.

2. Materials and Methods

2.1. Selection of Varieties and Cite of Experiment

Seed priming with thiamine and Indole-3-acetic acid (IAA) against different arsenic stress levels i.e., 0, 50 and 100 mg/kg sodium arsenite (NaAsO₂), was studied in two contrasting maize varieties Akbar and Pearl. The soil filled pot experiment was conducted in New Botanical Garden Government College University Faisalabad. Two varieties (Akbar and Pearl) were selected on the basis of having different As stress tolerance capacities in our previous studies (Atif and Perveen 2021 [55]). Akbar was found to be sensitive, while Pearl exhibited tolerance against As stress. Seeds were collected from the Maize and Millet Research Institute, Yousafwala, Sahiwal, Pakistan (MMRI).

2.2. Soil Analysis

In dry soil analysis, the soil had 0.81% organic matter, a saturation percentage of 32%, 6.3 mg kg⁻¹ NO₃-N, 2.99 mg kg⁻¹ NH₄-N, 188 mg kg⁻¹ K, 106 mg kg⁻¹ Ca, 5.11 mg kg⁻¹ available P, 2.3 dSm⁻¹ electrical conductivity (ECe) and 8.1 pH. The texture of the soil was sandy clay loam soil with 68% sand contents, 22% clay contents and 13% silt. Soil pH, inorganic nutrients of the soil saturated extract, soil saturation and ECe were determined with the described protocol by Jackson (1969) [56].

2.3. Experimental Lay Out

All the pots were arranged in a Completely Randomized Design (CRD) with three replicates of each treatment for each variety. The primed seeds were sown in plastic pots filled with 8 kg of soil. There were a total of 72 plastic pots (diameter 30 cm) (Arsenic stress (0, 50, 100 mg/kg)), (priming of seeds with thiamine (250 mg/L), priming of seeds with IAA (30 µM), priming of seeds with thiamine (250 mg/L) + IAA (30 µM) varieties (2)). The designated breakup of the pot experiment for each of variety was as follows: (1) 0 mg/kg As (0 mg/L thiamine + 0 µM IAA), (2) 0 mg/kg As (250 mg/L thiamine + 0 µM IAA), (3) 0 mg/kg As (0 mg/L thiamine + 30 µM IAA), (4) 0 mg/kg As (250 mg/L thiamine + 30 µM IAA), (5) 50 mg/kg As (0 mg/L thiamine + 0 µM IAA), (6) 50 mg/kg As (250 mg/L thiamine + 0 µM IAA), (7) 50 mg/kg As (0 mg/L thiamine + 30 µM IAA), (8) 50 mg/kg As (thiamine 250 mg/L + IAA 30 µM), (9) As mg/kg (thiamine 0 mg/L + IAA 0 µM), (10) 100 mg/kg As (250 mg/L thiamine + 0 µM IAA), (11) 100 mg/kg As (0 mg/L thiamine + 30 µM IAA), (12) 100 mg/kg As (250 mg/L thiamine + 30 µM IAA).

2.4. Seed Priming with Thiamine and IAA

Surface sterilization of seeds of both varieties with 5% hypochlorite solution for 10 min was carried out with distilled water. For seed priming, respective solutions of thiamine (250 mg/L) and IAA (30 µM) were prepared, and seeds were dipped in solutions of different treatments, i.e., control (non-primed seeds), thiamine (250 mg/L), IAA (30 µM) and a mixture of thiamine (250 mg/L) + IAA (30 µM) for 12 h. Then, seeds were air dried for 6 h at room temperature, and 8 healthy seeds were selected of each treatment and sown in soil filled plastic pots.

2.5. Arsenic Stress Applications

After two weeks of germination, the maize plants were subjected to different As stress levels (i.e., 0, 50 and 100 mg/kg). For this purpose, the solutions of different levels of As (50, 100 mg/kg) were prepared by dissolving NaAsO₂ with distilled water, and they were applied to each pot with the respective As level.

2.6. Statistical Analysis

A Microsoft excel sheet was used for data compilation and the calculation of mean values and standard errors. The application of a three-way ANOVA was performed to determine the differences at significance levels (p < 0.05) and the least significance difference test (LSD) by using the Co-Stat Computer Program (window version 6.303, PMB 320, Monterey, CA, USA). Among three factors, variety was the first factor; stress (arsenic in this case) was the 2nd factor, and all priming treatments, i.e., control (no priming), thiamine (250 ppm), IAA (30 µM) and a mixture of thiamine (250 ppm) + IAA (30 µM), were considered as the 3rd factor. Principal component analysis (PCA) and correlations were drawn by using R-studio.

2.7. Data Collection

The data were collected on 6-week-old maize plants for growth, photosynthetic attributes, gas exchange characters, plant water relation attributes, antioxidants and nutrient ions.

3. Growth Characteristics and Chlorophyll Attributes

The growth traits (root and shoot length) and biomass factors (root and shoot fresh and dry weight) of 6-week-old maize plants were determined. After that, samples were kept in an oven having a temperature of 65 °C for 7 days to calculate the dry weight of maize plants.

Photosynthetic attributes were appraised with the method of Arnon (1949) [57]. At 4 °C, 0.5 g of the leaf sample was homogenized in 80% acetone and then centrifuged for 5 min at 12,000× g rpm. The OD was determined at 480, 645 and 663 nm.

3.1. Leaf Gas Exchange and Plant Water Relation Attributes

The fully expanded leaf was used for gas exchange attributes such as the net rate of photosynthesis (A), transpiration rate (E), stomatal conductance (g_s) with a portable Infra-Red Gas Analyzer (Analytical Development Company, London, UK).

The method of Scholander et al. (1965) [58] was adapted to measure leaf water potential (Ψ_w). The excised leaf was subjected to a pressure chamber to measure the leaf water potential (Ψ_w). The same leaf was placed in a freezer at −40 °C and then thawed, and cell sap was extracted and used for the determination of osmotic potential (Ψ_s) with an osmometer (Wescor-5500, Wescor, Logan, UT, USA).

3.2. Malondialdehyde (MDA)

The fresh maize leaf (0.25 g) was grinded with liquid nitrogen, and then the sample was homogenized with 6% trichloroacetic acid (5 mL) and centrifuged at 10,000× g rpm for 10 min, and the supernatant was collected. After that, 0.5 mL of the sample was mixed with 5% thiobarbituric acid (TBA), and then the mixture was heated in a water bath at 95 °C. With the help of a spectrophotometer, OD was measured at 532 and 600 nm (Cakmak and Horst 1991 [59]).

3.3. Hydrogen Peroxide (H₂O₂)

In the case of H₂O₂, 0.5 mL of the supernatant was mixed with 1 M KI and 50 mM potassium phosphate buffer with a pH of 7.5 and incubated for 50 min, and the reading was taken at 390 nm (Velikova et al., 2000 [60]).

3.4. Enzyme Assays

The fresh maize leaf material was grinded with liquid nitrogen, and 10 mL of potassium phosphate buffer were added, and the supernatant was obtained after centrifugation at 10,000× g rpm for 20 min at 4 °C. For the enzymes’ assay, the supernatant was stored at −80 °C.

At 290 nm for 120 s, APX was measured with the protocol in the method by Nakano and Asada (1981) [61]. The reaction mixture contained (50 mM) phosphate buffer (pH 7.0), ascorbate (0.5 mM), EDTA (0.1 mM) and an enzyme extract (0.2 mL). At 560 nm, SOD activity was measured by using the procedure by Giannopolitis and Ries (1977) [62]. In the presence of the enzyme, photochemical inhibition of nitroblue tetrazolium chloride was followed to determine SOD at 560 nm. The reaction mixture comprised 50 mM phosphate buffer (pH 7.8), riboflavin (1.3 µM), EDTA (75 nM), methionine (13 mM), enzyme extract (50 µL).

POD activity was measured by following the protocol by Polle et al. (1994) [63]. The reaction mixture of 3 mL contained 100 µL enzyme, 20 mM guaiacol and 10 mM H₂O₂, and it was prepared and used to determine POD for 120 s at 470 nm. Catalase activity was measured with the method set by Chance and Maehly (1955) [64]. The reaction solution consists of 0.5 M of phosphate buffer (pH 7.0), 20 mM H₂O₂ and 0.1 mL of the enzyme extract. Optical density was monitored at 240 nm for 3 min with a spectrophotometer.

3.5. Total Phenolic and Flavonoid Contents

Phenolic contents were measured with the method by Wolfe et al. (2003) [65]. The fresh maize leaf was homogenized with 10 mL of 80% methanol and then centrifuged at 10,000× g rpm for 10 min at 4 °C. A Folin-Ciocalteu reagent was added in the supernatant with 20% Na₂CO₃, and at 750 nm, absorbance was taken out.

The flavonoids were determined with a spectrophotometer followed by the method by Marinova et al. (2005) [66]. Fresh leaf (0.5 g) was extracted in 80% ethanol. Then, 1 mL of extract was mixed with 300 µL AlCl₃ and 300 µL NaNO₂. The mixture was incubated at room temperature for 5 min. Then, 2 mL of 1 M NaOH were added in the mixture, and the volume was raised up to 10 mL with distilled water. Absorbance was taken at 510 nm.

3.6. Ascorbic Acid (AsA) and Total Soluble Proteins

The ascorbic acid was determined with the method by Mukherjee and Choudhuri et al. (1983) [67]. Fresh maize leaf (0.5 g) was homogenized with 10 mL of 6% TCA. After filtering of the homogenate, 2% dinitrophenyl hydrazine with one drop of 10% thiourea was added. The incubation of the reaction mixture was carried out at 95 °C for 40 min. An addition of 80% H₂SO₄ was made to the reaction mixture, and OD was read at 530 nm.

The supernatant that was used previously for the antioxidant enzymes’ assay was reacted with Bradford, and the reading was taken at 595 nm by using the approach of Bradford (1976) [68].

3.7. Total Soluble Sugars (TSS), Reducing Sugars (RS) and Non-Reducing Sugars (NRS)

The total soluble sugars were determined by following the Dubois et al., 1956 [69] protocol. The fresh maize leaf samples were reacted with an anthrone reagent, and at 490 nm, absorbance was taken. The reducing sugars were determined with the protocol by Henson and Stone (1988) [70]. The difference between TSS and RS determined the non-reducing sugar.

3.8. Total Free Amino Acids (TFAA) and Proline

The TFAA contents were determined with the described protocol by Hamilton and Van Slyke (1943) [71]. The 1 mL extract that was used in the enzyme assay was taken and then mixed with 1 mL of 10% pyridine and the same with 2% ninhydrin. After that, the heated mixture for 30 min at 95 °C was used for absorbance at 570 nm.

Leaf free proline was determined with the described protocol by Bates et al. (1973) [72]. The homogenate fresh maize leaf with 3% sulphosalycylic acid was filtered, and the volume was maintained at equal to the volume of ninhydrin after filtration. The 5 mL of toluene was added, and the mixture was incubated at 95 °C for 45 min, and then absorbance was read at 520 nm.

3.9. Anthocyanins

Anthocyanin contents were measured with the protocol described by Mirecki and Teramura (1984) [73]. Fresh leaf (0.2 g) was homogenized with 2 mL of 1% methanol (acidified). Then, the mixture was centrifuged at 12,000× g rpm for 20 min, and the reading was taken at 530 nm, 657 nm, respectively, with the help of the spectrophotometer.

4. Nutritive Ion Analysis and As Determination

Determination of Arsenic, K⁺, Ca²⁺, P

Plant dry material (0.1 g) was used for individual ion determination of the root and shoot (Allen et al., 1986 [74]). The dry plant sample (0.1 g) was digested with concentrated H₂SO₄ in a digestion flask. After incubation for 24 h, the mixture was heated at 150 °C with the addition of 35% H₂O₂. The repetition of the same step continued until the transparent solution was obtained.

The total As contents were determined with an atomic absorption spectrophotometer (novA A 400, Analytik Jena, Jena, Germany). Potassium (K⁺) and calcium (Ca²⁺) contents were measured with a flame photometer (Sherwood, Model 360, Cambridge, UK). Phosphorous (P) contents were determined by using the method outlined by Jackson (1969) [56].

5. Results

5.1. Growth Attributes and Chlorophyll Contents

The growth-related characteristics (root and shoot length, fresh and dry biomasses) of both varieties underwent a significant decrease under both As-stressed levels (50, 100 mg/kg) (

Table 1. Shoot fresh and dry weights, root fresh and dry weights by exogenously applied thiamine (250 mg/L) and IAA (30 µM) as priming agent of two maize (Zea mays L.) varieties grown under control (C) and arsenic stressed (50, 100 mg/kg) conditions (Mean ± SE; n = 3).

	SFW (g)			SDW (g)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	20.47 ± 0.47 a	15.63 ± 0.39 d	13.44 ± 0.56 b	1.37 ± 0.02 c	1.09 ± 0.01 d	0.97 ± 0.01 d
Thia. 250 mg/L	20.24 ± 0.58 a	16.51 ± 0.63 c	13.74 ± 0.27 b	1.50 ± 0.02 a	1.19 ± 0.01 c	1.16 ± 0.01 c
IAA 30 µM	17.40 ± 0.49 c	17.33 ± 0.58 b	13.37 ± 0.40 b	1.41 ± 0.01 b	1.24 ± 0.01 b	1.30 ± 0.02 b
IAA 30 µM + Thia 250 mg/L	19.48 ± 0.53 b	19.48 ± 0.52 a	14.64 ± 0.47 a	1.49 ± 0.01 a	1.29 ± 0.01 a	1.39 ± 0.02 a
Pearl
Control	16.44 ± 0.57 c	12.60 ± 0.38 d	12.60 ± 0.42 d	1.61 ± 0.02 d	1.18 ± 0.03 d	1.15 ± 0.03 d
Thia. 250 mg/L	17.54 ± 0.37 b	14.42 ± 0.46 b	14.37 ± 0.54 b	1.77 ± 0.03 c	1.40 ± 0.03 c	1.42 ± 0.03 c
IAA 30 µM	16.03 ± 0.24 c	13.53 ± 0.61 c	13.26 ± 0.50 c	1.94 ± 0.01 b	1.63 ± 0.02 b	1.63 ± 0.02 b
IAA 30 µM + Thia 250 mg/L	18.33 ± 0.41 a	16.77 ± 0.55 a	15.70 ± 0.62 a	2.03 ± 0.03 a	1.76 ± 0.02 a	1.71 ± 0.01 a
LSD 5%	0.57			0.02
	RFW (g)			RDW (g)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	2.55 ± 0.11 d	2.11 ± 0.03 c	1.89 ± 0.03 d	0.28 ± 0.006 d	0.17 ± 0.006 d	0.11 ± 0.009 d
Thia. 250 mg/L	2.85 ± 0.09 b	2.41 ± 0.05 b	2.35 ± 0.04 b	0.31 ± 0.006 b	0.26 ± 0.006 b	0.17 ± 0.006 c
IAA 30 µM	2.64 ± 0.05 c	2.35 ± 0.04 b	2.23 ± 0.03 c	0.29 ± 0.006 c	0.25 ± 0.006 c	0.27 ± 0.017 b
IAA 30 µM + Thia 250 mg/L	3.00 ± 0.07 a	2.54 ± 0.05 a	2.43 ± 0.03 a	0.34 ± 0.006 a	0.30 ± 0.009 a	0.35 ± 0.012 a
Pearl
Control	2.20 ± 0.06 d	1.98 ± 0.06 c	2.00 ± 0.11 c	0.33 ± 0.011 d	0.19 ± 0.009 d	0.15 ± 0.006 d
Thia. 250 mg/L	2.52 ± 0.05 b	2.30 ± 0.05 a	2.06 ± 0.03 bc	0.42 ± 0.009 b	0.25 ± 0.006 c	0.22 ± 0.009 c
IAA 30 µM	2.42 ± 0.05 c	2.24 ± 0.06 ab	2.09 ± 0.06 b	0.38 ± 0.006 c	0.31 ± 0.014 b	0.29 ± 0.011 b
IAA 30 µM + Thia 250 mg/L	2.76 ± 0.04 a	2.23 ± 0.06 b	2.28 ± 0.03 a	0.45 ± 0.006 a	0.41 ± 0.014 a	0.37 ± 0.009 a
LSD 5%	0.07			0.01

Means in a column with same letter (a, b, c,…) do not differ significantly.

and

Table 2. Shoot length, root length, Chl a, chl b by exogenously applied thiamine (250 mg/L) and IAA (30 µM) as priming agent of two maize (Zea mays L.) varieties grown under control (C) and arsenic stressed (50, 100 mg/kg) conditions (Mean ± SE; n = 3).

	SL (cm)			RL (cm)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	90.03 ± 1.05 b	74.77 ± 1.10 c	59.93 ± 0.89 d	19.47 ± 0.41 d	15.73 ± 0.46 c	13.47 ± 0.48 d
Thia. 250 mg/L	92.17 ± 1.50 a	85.53 ± 0.66 b	82.93 ± 0.95 b	21.30 ± 0.55 b	18.43 ± 0.57 a	15.83 ± 0.23 b
IAA 30 µM	90.87 ± 0.58 b	86.03 ± 0.90 b	81.67 ± 0.55 c	20.47 ± 0.67 c	17.47 ± 0.41 b	14.80 ± 0.55 c
IAA 30 µM + Thia 250 mg/L	92.87 ± 0.82 a	89.17 ± 0.46 a	84.70 ± 0.84 a	23.37 ± 0.54 a	18.73 ± 0.56 a	16.80 ± 0.55 a
Pearl
Control	94.03 ± 0.77 c	83.50 ± 2.84 c	84.33 ± 0.63 c	20.80 ± 0.68 c	18.37 ± 0.66 c	14.90 ± 0.26 d
Thia. 250 mg/L	95.63 ± 0.66 b	88.47 ± 0.56 b	87.57 ± 0.50 b	23.53 ± 0.66 b	21.40 ± 0.66 a	19.27 ± 0.43 b
IAA 30 µM	96.63 ± 0.69 ab	89.33 ± 0.56 b	87.33 ± 0.82 b	20.50 ± 0.65 c	19.50 ± 0.70 b	17.63 ± 0.63 c
IAA 30 µM + Thia 250 mg/L	97.10 ± 0.96 a	92.70 ± 0.94 a	91.47 ± 0.56 a	24.47 ± 0.52 a	20.80 ± 0.58 a	20.80 ± 0.76 a
LSD 5%	1.15			0.66
	Chl a (mg g−1 FW)			Chl b (mg g−1 FW)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	1.07 ± 0.03 d	0.67 ± 0.01 d	0.57 ± 0.01 d	0.32 ± 0.02 a	0.30 ± 0.01 a	0.30 ± 0.01 a
Thia. 250 mg/L	1.28 ± 0.02 b	0.93 ± 0.02 b	0.67 ± 0.01 b	0.30 ± 0.01 c	0.26 ± 0.01 c	0.23 ± 0.01 b
IAA 30 µM	1.17 ± 0.00 c	0.82 ± 0.01 c	0.61 ± 0.02 c	0.32 ± 0.00 a	0.19 ± 0.01 d	0.22 ± 0.01 c
IAA 30 µM + Thia 250 mg/L	1.36 ± 0.01 a	0.99 ± 0.01 a	0.79 ± 0.01 a	0.31 ± 0.01 b	0.28 ± 0.01 b	0.23 ± 0.01 b
Pearl
Control	1.05 ± 0.01 d	0.57 ± 0.00 c	0.45 ± 0.00 d	0.34 ± 0.01 b	0.31 ± 0.00 c	0.26 ± 0.01 d
Thia. 250 mg/L	1.15 ± 0.01 b	0.64 ± 0.00 b	0.57 ± 0.00 b	0.36 ± 0.00 a	0.35 ± 0.00 b	0.33 ± 0.01 a
IAA 30 µM	1.08 ± 0.01 c	0.64 ± 0.00 b	0.51 ± 0.01 c	0.36 ± 0.01 a	0.30 ± 0.01 d	0.31 ± 0.01 b
IAA 30 µM+ Thia 250 mg/L	1.23 ± 0.04 a	0.79 ± 0.01 a	0.71 ± 0.01 a	0.34 ± 0.01 b	0.36 ± 0.01 a	0.29 ± 0.01 c
LSD 5%	0.02			0.01

Means in a column with same letter (a, b, c,…) do not differ significantly.

). Overall, Akbar exposed more damage than Pearl. A total of 100 mg/kg of As-stress decreased SFW by 34.35% and 28.22%, RFW by 25.59% and 9.09%, SL by 33.43% and 10.31%, RL by 30.83% and 28.36%, SDW by 29.92% and 28.57%, and RDW by 64.28% and 54.54%, respectively, of Akbar and Pearl as compared to the control. However, pre-sowing treated maize seeds with thiamine (250 ppm) and IAA (30 μM) significantly enhanced growth traits, and a maximum increase was prominent in both maize varieties, pointedly in Pearl with combined and alone treated seeds under the control and stressed conditions. Among singly applied treatments, thiamine improved better than IAA. The maximum recovery trend was noted with combined treated seeds with Thiamine + IAA. Combined treatment of both increased the SFW (7.14, 24.6%), RFW (28, 14%), SL (41.16, 8.45%), RL (24.81, 39.59%), SDW (44.79, 49%) and RDW (40, 46.66%), respectively, in Akbar and Pearl under 100 ppm of As-stress (Table 1 and Table 2).

A significant decline in chlorophyll characters (a, b, a + b, carotenoids, Chl ratio) was concluded in both varieties, but Pearl was superior to Akbar (Table 2 and

Table 3. Chl a/b, Chl a + b, carotenoids, transpiration rate by exogenously applied thiamine (250 mg/L) and IAA (30 µM) as priming agent of two maize (Zea mays L.) varieties grown under control (C) and arsenic stressed (50, 100 mg/kg) conditions (Mean ± SE; n = 3).

	Chl a/b			Chl a + b (mg g−1 FW)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	3.38 ± 0.29 c	2.24 ± 0.14 c	1.94 ± 0.12 c	1.39 ± 0.01 d	0.96 ± 0.00 d	0.87 ± 0.00 c
Thia. 250 mg/L	4.34 ± 0.24 a	3.56 ± 0.24 b	2.96 ± 0.17 b	1.58 ± 0.01 b	1.20 ± 0.01 b	0.89 ± 0.00 b
IAA 30 µM	3.68 ± 0.08 b	3.91 ± 0.48 a	2.79 ± 0.23 b	1.49 ± 0.00 c	1.04 ± 0.04 c	0.83 ± 0.00 d
IAA 30 µM + Thia 250 mg/L	4.43 ± 0.19 a	3.50 ± 0.11 b	3.49 ± 0.20 a	1.67 ± 0.00 a	1.28 ± 0.00 a	1.02 ± 0.01 a
Pearl
Control	3.06 ± 0.08 bc	1.83 ± 0.05 b	1.72 ± 0.01 b	1.39 ± 0.00 d	0.88 ± 0.00 d	0.71 ± 0.01 d
Thia. 250 mg/L	3.20 ± 0.03 b	1.83 ± 0.01 b	1.80 ± 0.05 b	1.51 ± 0.01 b	1.00 ± 0.01 b	0.88 ± 0.00 b
IAA 30 µM	2.96 ± 0.08 c	2.12 ± 0.04 a	1.84 ± 0.02 b	1.44 ± 0.00 c	0.94 ± 0.01 c	0.82 ± 0.00 c
IAA 30 µM + Thia 250 mg/L	3.69 ± 0.31 a	2.20 ± 0.10 a	2.43 ± 0.11 a	1.57 ± 0.02 a	1.15 ± 0.00 a	1.00 ± 0.00 a
LSD 5%	0.21			0.01
	Carotenoids (mg g−1 FW)			Trans rate (µmol m−2 s−1)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	0.014 ± 0.000 a	0.012 ± 0.000 a	0.009 ± 8.89E a	0.43 ± 0.01 d	0.32 ± 0.01 d	0.25 ± 0.01 d
Thia. 250 mg/L	0.015 ± 0.000 a	0.013 ± 3.43E a	0.012 ± 4.29E a	0.47 ± 0.00 c	0.35 ± 0.00 c	0.29 ± 0.01 c
IAA 30 µM	0.014 ± 0.000 a	0.014 ± 0.001 a	0.010 ± 6.44E a	0.55 ± 0.00 b	0.38 ± 0.01 b	0.34 ± 0.01 b
IAA 30 µM + Thia 250 mg/L	0.015 ± 0.000 a	0.013 ± 5.73E a	0.013 ± 0.000 a	0.62 ± 0.01 a	0.42 ± 0.01 a	0.37 ± 0.01 a
Pearl
Control	0.011 ± 0.000 a	0.011 ± 5.72E a	0.010 ± 0.000 a	0.45 ± 0.01 d	0.38 ± 0.01 d	0.33 ± 0.00 d
Thia. 250 mg/L	0.012 ± 0.000 a	0.011 ± 0.000 a	0.011 ± 0.000 a	0.46 ± 0.00 c	0.42 ± 0.01 c	0.41 ± 0.01 c
IAA 30 µM	0.011 ± 0.000 a	0.014 ± 0.000 a	0.010 ± 0.000 a	0.51 ± 0.00 b	0.43 ± 0.00 b	0.45 ± 0.00 b
IAA 30 µM + Thia 250 mg/L	0.013 ± 0.000 a	0.014 ± 0.000 a	0.013 ± 4.6E a	0.59 ± 0.01 a	0.47 ± 0.00 a	0.46 ± 0.01 a
LSD 5%	3.42			0.01

Means in a column with same letter (a, b, c,…) do not differ significantly.

). The highest As-stress level decreased Chl. a (42.73, 42.66%), total Chl (48.92, 37.41%) and carotenoids (38.46, 18.18%), respectively, in Akbar and Pearl as compared to the control. Thiamine and IAA pre-sowing treatments as alone and in combination established resistance against chlorophyll degradation in both maize varieties. The priming treatments of seeds with the combination proved to be better than the individuals. Singly applied thiamine efficiently improved Chl attributes more so than IAA. The combined treated plants increased Chl. a (38.59, 57.77%), total Chl (14.94, 40.84%) and carotenoids (50, 33.33%), respectively, in Akbar and Pearl against 100 ppm of As-stress (Table 3).

5.2. Leaf Gas Exchange Attributes

A significant reduction was confirmed in the leaf gas exchange machinery, i.e., the net rate of photosynthesis (A), transpiration rate (E) and stomatal conductance (g_s) were observed in both maize varieties under As-stress in the current study (Figure 1A–C). A significantly decreased under both As-stressed levels (50 and 100 mg/kg) in both varieties, but Akbar was affected more severely than Pearl (Figure 1A). A total of 100 mg/kg of As-stress reduced the NRP (32.58, 19.58%) of Akbar and Pearl, respectively, as compared to the control. However, a remarkable increase in A was noticed under pre-sowing treated maize seeds with thiamine and IAA as alone and in the mixture under in both maize varieties in the stressed and non-stressed conditions. Among alone treatments, thiamine proved to be better than IAA. However, both varieties showed a maximum improvement with treated seeds of thiamine and IAA as combined under both As-stressed levels (Figure 1A). A combined treatment of both thiamine and IAA increased A (33.33, 23.07%) of Akbar and Pearl under 100 mg/kg of As-stress. The transpiration rate (E) significantly decreased under both levels (50 and 100 mg/kg) of As-stress in both maize varieties, but Akbar presented the most decrement in this variable (Table 3). Of the two As levels, 100 mg/kg As reduced E (53.48, 28.26%) of Akbar and Pearl, respectively, as compared to the control. However, IAA and thiamine treated maize seeds singly and in the mixture significantly improved, and a maximum increase was manifested as treated in the mixture of thiamine and IAA under both As-stressed levels (50 and 100 mg/kg) in both maize varieties. A combined treatment of both increased E (54.18, 39.40%) of Akbar and Pearl under 100 ppm of As-stress. Stomatal conductance (g_s) significantly decreased under both As-stressed levels (50 and 100 mg/kg) in both varieties. A total of 100 mg/kg of As-stress decreased g_s (23.65, 14.13%) of Akbar and Pearl, respectively, compared to the control. Both varieties manifested a maximum increase with combined treated seeds under As-stressed conditions. A combined treatment of both thiamine and IAA increased (g_s) (23.94, 19%) of Akbar and Pearl under 100 mg/kg of As-stress (Figure 1B).

5.3. Plant Water Relation

Water potential (Ψ_s) decreased against both levels (50 and 100 mg/kg) of As-stress in both maize varieties, but a maximum decrease was observed in Akbar (Figure 1C). A total of 100 mg/kg of As-stress decreased Ψ_s (61.69, 30.61%), respectively, in Akbar and Pearl as compared to the control. However, IAA and thiamine treated seeds as a mixture and alone improved significantly under both levels (50 and 100 mg/kg) of As-stress (Figure 1C). Primed seeds with thiamine alone performed better than IAA. Ψ_s increased by 52% and 80% with the combined treatment under 100 ppm of As-stress in Akbar and Pearl, respectively. Ψ_w was significantly disturbed under both As-stressed levels (50 and 100 mg/kg) of both varieties. A total fo 100 ppm of As-stress decreased Ψ_w (40.16, 13.88%), respectively, in Akbar and Pearl as compared to the control. However, IAA and thiamine treated seeds significantly improved Ψ_w when applied singly and in a mixture, but a maximum increase was noticed under a combination of IAA and thiamine (Figure 1D). Of all treatments, the combined treatment of both thiamine and IAA improved the maximum, while among singly applied treatments, thiamine improved better. Ψ_w increased by 53.42% and 8.11% with the combined treatment under 100 mg/kg of As stress in Akbar and Pearl, respectively.

5.4. Activities of Antioxidant Enzymes

A significant increase in POD, SOD, CAT and APX activities was recorded in both maize varieties under both As-stressed levels (50 and 100 mg/kg) (Figure 2A–D). A total of 100 mg/kg of As-stress increased POD (50, 57.23%), SOD (16.33, 20.46%), CAT (51.44, 53.29%) and APX (44.72, 50.07%) activities of Akbar and Pearl, respectively, as compared to the control. The exogenously applied thiamine and IAA pre-sowing treatments of maize seeds as individuals and in combination induced significant improvements. However, a maximum decrease was recorded with the combined treatment of both under both stressed levels. The combined treatment decreased POD (45.54, 22.7%), CAT (18.06, 18.11%) and APX (29.66, 22.17%) activities of Akbar and Pearl, respectively, under 100 mg/kg of As-stress.

5.5. Non-Enzymatic Antioxidants

The decrease in phenolic contents while the increase in flavonoid, ASA and anthocyanin was recorded under both As-stress levels (50 and 100 mg/kg) of both varieties (Figure 3A–D). Phenolic contents decreased by 32.07% and 35.8% in Akbar and Pearl at 100 mg/kg of As-stress. The thiamine and IAA as individuals and as the combined pre-sowing treatments of maize seeds in As-contaminated conditions clearly enhanced the phenolic compounds. A total of 100 mg/kg of As-stress increased ASA (19.45, 11.82%), anthocyanin (33.42, 42.18%) and flavonoids (108, 72.41%), respectively, in Akbar and Pearl as compared to the control. However, improvements in plants where thiamine and IAA as pre-sowing treated maize seeds were applied as combined under both As-stressed levels (50 and 100 mg/kg) were maximum. The combined treatment decreased ASA (14.94, 6.91%) and anthocyanin (16.16, 12.08%), while flavonoids (28.63, 30.67%) decreased with thiamine alone in Akbar and Pearl, respectively, under 100 mg/kg of As-stress.

5.6. Oxidative Stress Markers

The levels of H₂O₂ and MDA increased against diverse As-stressed levels (50 and 100 mg/kg) and revealed a significant increase in both varieties (

Table 4. H₂O₂, MDA, TSP, FAA by exogenously applied thiamine (250 mg/L) and IAA (30 µM) priming agent of two maize (Zea mays L.) varieties grown under control (C) and arsenic stressed (50, 100 mg/kg) conditions (Mean ± SE; n = 3).

	H2O2 (µmolg−1 FW)			MDA (nmolg−1 FW)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	11.28 ± 0.07 a	15.03 ± 0.11 a	15.93 ± 0.08 a	7.36 ± 0.82 c	8.95 ± 0.36 a	11.67 ± 0.42 a
Thia. 250 mg/L	10.32 ± 0.11 c	13.61 ± 0.09 c	14.73 ± 0.39 c	8.68 ± 0.60 b	9.13 ± 0.66 a	10.16 ± 0.61 b
IAA 30 µM	10.64 ± 0.10 b	13.96 ± 0.07 b	15.21 ± 0.12 b	9.87 ± 0.59 a	7.65 ± 0.30 b	9.58 ± 0.30 b
IAA 30 µM + Thia 250 mg/L	9.96 ± 0.11 d	13.06 ± 0.12 d	14.02 ± 0.07 d	5.09 ± 0.66 d	6.10 ± 1.15 c	8.91 ± 0.27 c
Pearl
Control	10.78 ± 0.10 a	13.10 ± 0.11 a	14.76 ± 0.09 a	7.81 ± 0.32 a	8.43 ± 0.10 b	10.12 ± 0.68 a
Thia. 250 mg/L	10.27 ± 0.07 c	12.51 ± 0.06 c	14.17 ± 0.10 c	8.28 ± 0.57 a	8.70 ± 0.58 b	8.41 ± 0.54 bc
IAA 30 µM	10.48 ± 0.08 b	12.85 ± 0.06 b	14.39 ± 0.09 b	7.11 ± 0.16 b	9.47 ± 0.59 a	8.14 ± 0.20 c
IAA 30 µM + Thia 250 mg/L	10.09 ± 0.11 d	11.97 ± 0.08 d	13.39 ± 0.09 d	5.77 ± 0.73 c	8.28 ± 0.39 b	8.88 ± 0.71 b
LSD 5%	0.14			0.66
	TSP (mg g−1 FW)			TFAA (mg g−1 FW)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	536.46 ± 4.80 c	488.13 ± 4.76 a	404.14 ± 5.66 d	7.07 ± 0.44 a	11.03 ± 0.44 b	15.86 ± 0.15 a
Thia. 250 mg/L	588.91 ± 4.76 a	490.53 ± 6.28 a	479.56 ± 5.64 b	4.54 ± 0.06 b	4.32 ± 0.07 d	12.74 ± 0.16 b
IAA 30 µM	517.26 ± 4.75 d	479.90 ± 5.12 b	522.06 ± 4.80 a	4.25 ± 0.08 c	5.49 ± 0.07 c	11.69 ± 0.15 c
IAA 30 µM + Thia 250 mg/L	560.46 ± 4.71 b	471.67 ± 5.95 c	438.08 ± 4.48 c	4.52 ± 0.10 b	14.78 ± 0.20 a	10.85 ± 0.11 d
Pearl
Control	575.54 ± 5.05 b	511.44 ± 5.39 d	493.61 ± 7.99 d	7.64 ± 0.29 c	14.71 ± 0.11 a	14.66 ± 0.17 a
Thia. 250 mg/L	610.84 ± 5.66 a	562.51 ± 4.75 b	537.15 ± 10.42 c	13.61 ± 0.12 a	13.69 ± 0.15 b	13.55 ± 0.14 b
IAA 30 µM	571.08 ± 4.38 b	540.23 ± 4.53 c	570.74 ± 4.28 b	13.63 ± 0.13 a	14.59 ± 0.10 a	14.56 ± 0.07 a
IAA 30 µM + Thia mg/L	615.99 ± 3.89 a	606.73 ± 8.37 a	591.65 ± 3.86 a	12.90 ± 0.22 b	14.72 ± 0.10 a	13.12 ± 0.10 c
LSD 5%	6.47			0.21

Means in a column with same letter (a, b, c,…) do not differ significantly.

). A total of 100 ppm of As-stress increased H₂O₂ (41.08, 36.94%) and MDA (58.63, 29.61%), respectively, in Akbar and Pearl as compared to the control. Thiamine and IAA pre-sowing treatments of maize seeds improved these significantly in both varieties under both As-stressed levels (50 and 100 mg/kg) as individuals and combined. The maximum decrease was observed with thiamine and IAA as combined. The combined treatment decreased H₂O₂ (10.44, 13.33%), MDA (12.4, 19.48%) in Akbar and Pearl, respectively, under 100 mg/kg of As-stress (Table 4).

5.7. TSP, TFAA

The exposure of maize plants to As-stress resulted in a marked decrease in total soluble proteins (TSP) (Table 2). A total of 100 mg/kg of As-stress decreased TSP 24.66% and 15% in Akbar and Pearl compared to the control. Total free amino acids (TFAA) also increased in maize plants under both As-stressed levels (50 and 100 mg/kg) (Table 4). A total fo 100 ppm As-stress increased TFAA (124.18, 91.75%) in Akbar and Pearl compared to the control. The impact of exogenous application of thiamine and IAA as pre-sowing treated maize seeds was significant as recorded in TSP and TFAA under both As-stressed levels (50 and 100 mg/kg) (Table 4).

5.8. Proline TSS, RS, NRS

Under both As-stressed levels (50 and 100 mg/kg), a significant increase in proline and TSS was recorded in both maize varieties (

Table 5. TSS, reducing sugars, non-reducing sugars and proline by exogenously applied thiamine (250 ppm) and IAA (30 µM) as priming agent of two maize (Zea mays L.) varieties grown under control (C) and arsenic stressed (50, 100 mg/kg) conditions (Mean ± SE; n = 3).

	TSS (mg g−1 FW)			Reducing sugars (mg g−1 FW)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	90.13 ± 0.87 d	142.15 ± 0.94 a	149.42 ± 0.60 a	15.60 ± 1.53 d	11.57 ± 0.66 d	7.80 ± 0.25 d
Thia 250 mg/L	146.41 ± 2.00 b	115.47 ± 3.72 c	115.82 ± 17.73 c	23.39 ± 0.87 c	14.34 ± 0.87 c	10.56 ± 0.43 c
IAA 30 µM	128.80 ± 2.40 c	100.88 ± 1.96 d	131.07 ± 4.12 b	28.93 ± 0.91 b	19.12 ± 0.66 b	14.34 ± 0.87 b
IAA 30 µM+ Thia 250 mg/L	169.56 ± 2.63 a	130.31 ± 3.52 b	153.46 ± 2.55 a	31.70 ± 0.87 a	24.40 ± 0.66 a	17.61 ± 0.66 a
Pearl
Control	142.86 ± 0.90 d	166.90 ± 1.39 a	179.79 ± 0.74 a	22.14 ± 0.66 d	14.09 ± 0.66 d	9.81 ± 0.87 d
Thia 250 mg/L	171.06 ± 3.09 b	135.34 ± 3.70 c	110.69 ± 2.63 d	28.17 ± 1.53 c	20.38 ± 1.31 c	20.38 ± 1.74 c
IAA 30 µM	159.74 ± 1.65 c	122.01 ± 1.33 d	139.87 ± 3.49 c	35.72 ± 1.53 b	32.45 ± 0.87 b	30.94 ± 0.87 b
IAA 30 µM+ Thia 250 mg/L	177.86 ± 3.49 a	153.96 ± 2.86 b	146.91 ± 2.40 b	43.77 ± 1.57 a	38.24 ± 1.10 a	38.99 ± 1.10 a
LSD 5%	5.1			1.2
	Non reducing sugar (mg g−1 FW)			Proline (µmolg−1 FW)
	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg	As 0 mg/kg	As 50 mg/kg	As 100 mg/kg
Akbar
Control	129.05 ± 3.80 b	77.48 ± 1.00 d	64.65 ± 2.97 d	0.98 ± 0.01 a	1.21 ± 0.01 a	1.33 ± 0.01 a
Thia 250 mg/L	123.02 ± 2.65 c	101.13 ± 3.46 b	89.81 ± 2.00 c	0.96 ± 0.00 b	0.98 ± 0.01 b	1.10 ± 0.01 b
IAA 30 µM	99.87 ± 1.53 d	81.76 ± 2.63 c	116.73 ± 4.39 b	0.96 ± 0.01 b	0.98 ± 0.00 b	1.10 ± 0.01 b
IAA 30 µM+ Thia 250 mg/L	137.86 ± 1.76 a	105.91 ± 2.90 a	135.85 ± 2.86 a	0.95 ± 0.00 c	0.96 ± 0.00 c	1.07 ± 0.03 c
Pearl
Control	130.56 ± 3.46 b	100.63 ± 1.76 b	76.98 ± 3.14 c	0.90 ± 0.01 a	1.10 ± 0.00 a	1.21 ± 0.01 a
Thia 250 mg/L	142.89 ± 4.62 a	114.96 ± 4.53 a	90.31 ± 3.90 b	0.87 ± 0.00 b	1.11 ± 0.01 a	1.12 ± 0.01 b
IAA 30 µM	124.02 ± 2.97 c	89.56 ± 2.01 c	108.93 ± 3.21 a	0.90 ± 0.01 a	0.99 ± 0.00 b	1.09 ± 0.01 c
IAA 30 µM+ Thia 250 mg/L	134.09 ± 4.30 b	115.72 ± 2.51 a	107.92 ± 1.90 a	0.83 ± 0.00 c	0.98 ± 0.01 c	0.98 ± 0.00 d
LSD 5%	3.59			0.01

Means in a column with same letter (a, b, c,…) do not differ significantly.

). A total fo 100 mg/kg of As-stress increased proline (34.69, 34.83%) and TSS (65.78, 25.84%), respectively, in Akbar and Pearl compared to the control. Plants treated with thiamine and IAA as pre-sowing treated seeds decreased proline and TSS contents under both As-stressed levels (50 and 100 mg/kg). In contrast, the reducing sugar and non-reducing sugar contents decreased under both As-stressed levels (50 and 100 mg/kg) (Table 5). An increase in reducing sugars was observed with pre-sowing treated maize seeds with thiamine and IAA as combined and as alone under both As-stressed levels (50 and 100 mg/kg). However, the combined priming treatment as combined (thiamine + IAA) improved the maximum under both As-stress levels (Table 5).

5.9. Ion Uptake

A significant decrease in shoot and roots Ca²⁺, K⁺ and P of both maize varieties under As-stress was recorded (Figure 4A–D and Figure 5A–C;

Table 6. Mean squares from analysis of variance of the data for various growth, photosynthesis, gas exchange, osmolytes, antioxidants and nutrient ions attributes with seed priming of maize with thiamine and IAA under As-stress.

Source of Variation	Varieties (Var)	Stress (S)	Treatment (T)	Var × S	Var × T	S × T	Var × S × T	Error
Shoot f. wt.	50.601 ***	114.254 ***	19.963 ***	16.108 ***	2.353 *	4.465 ***	1.059 ns	0.731
Shoot dry wt.	1.820 ***	0.711 ***	0.496 ***	0.027 ***	0.076 ***	0.018***	0.006 ***	0.001
Root f. wt.	0.647 ***	1.346 ***	0.557 ***	0.042 *	0.014 ns	0.018 ns	0.022 ns	0.010
Root dry wt.	0.060 ***	0.077 ***	0.084 ***	0.005 ***	0.001 **	0.008***	0.002 ***	2.5 × 10−4
Shoot length	749.490 ***	777.910 ***	350.603 ***	69.033 ***	71.654 ***	54.546***	36.789 ***	2.932
Root length	85.151 ***	154.348 ***	48.803 ***	4.875 *	1.460 ns	1.997 ns	0.949 ns	0.961
Chl. a	0.304 ***	2.054 ***	0.205 ***	0.018 ***	0.006 ***	0.001*	0.003 ***	6.6 × 10−4
Chl. b	0.052 ***	0.023 ***	0.001 **	0.001 **	0.006 ***	0.003***	0.001 **	3.0× 10−4
Chlorophyll ratio	16.677 ***	9.854 ***	2.641 ***	0.567 **	0.913 ***	0.337**	0.072 ns	0.098
Total Chlorophyll	0.109 ***	2.499 ***	0.210 ***	0.011 ***	0.001 *	0.003***	0.006 ***	3.9× 10−4
Carotenoids	1.52 × 10−5 ***	3.55 × 10−5 ***	1.81 × 10−5 ***	1.3 × 10−5 ***	1.8 × 10−6 ***	3.63 × 10−6 ***	7.96 × 10−7 *	2.6 × 10−7
A	0.429 ***	0.520 ***	0.264 ***	0.049 ***	0.001 *	0.010***	0.008 ***	5.0× 10−4
E	0.042 ***	0.144 ***	0.055 ***	0.020 ***	4.38 × 10−4 ns	0.002***	4.61 × 10−4 *	1.7 × 10−4
gs	0.034 ***	0.175 ***	0.041 ***	0.081 ***	0.019 ***	0.007 ***	0.009 ***	2.0 × 10−4
Ψw	3.454 ***	2.398 ***	0.126 ***	0.696 ***	2.671 ns	0.005 ***	0.002 **	5.2 × 10−4
Ψs	0.256 ***	0.495 ***	0.411 ***	0.235 ***	0.226 ***	0.211 ***	0.058 ***	0.002
MDA	0.047 ns	38.618 ***	11.702 ***	6.976 **	1.902 ns	1.965ns	1.304 ns	1.009
H2O2	10.083 ***	105.028 ***	6.173 ***	2.049 ***	0.429 ***	0.145 **	0.023 ns	0.044
AsA	12.416 *	427.615 ***	88.657 ***	76.755 ***	15.739 ***	20.209 ***	14.825 ***	1.775
TSP	82,082.92 ***	30,318.97 ***	8005.24 ***	3120.6 ***	4467.6 ***	3953.3 ***	1262.33 ***	93.297
TFAA	367.610 ***	145.775 ***	9.897 ***	52.619 ***	35.337 ***	23.227 ***	13.585 ***	0.103
TSS	6817.01 ***	1336.84 ***	2451.77 ***	782.33 ***	650.06 ***	3066.6***	152.128 *	57.861
RS	1673.90 ***	614.41 ***	1241.20 ***	38.382 ***	131.00 ***	2.067 ns	18.416 ***	3.211
NRS	674.499 ***	6740.893 ***	2269.101 ***	646.75 ***	375.2 ***	1300.0 ***	202.300 ***	28.628
TPC	193.187 ***	2786.794 ***	2651.804 ***	42.611 ***	21.757 **	1054.8***	55.380 ***	3.421
TFC	36.528 ***	94.788 ***	438.796 ***	53.768 ***	64.258 ***	319.749***	23.996 ***	0.848
TAC	0.014 ***	0.087 ***	0.009 ***	0.014 ***	0.057 ***	0.027***	0.008 ***	2.2 × 10−4
Proline	0.033 ***	0.251 ***	0.080 ***	0.014 ***	0.013 ***	0.012***	0.004 ***	3.4 × 10−4
CAT	3.666 **	65.547 ***	6.367 ***	4.934 ***	1.086 *	2.488***	2.462 ***	0.324
POD	664.127 ***	712.353 ***	137.162 ***	78.399 ***	0.835 ns	3.221**	4.579 ***	0.506
SOD	1352.80 ***	2872.00 ***	274.408 ***	2500.6 ***	155.86 ***	522.25 ***	184.085 ***	0.983
APX	52.833 ***	44.595 ***	17.466 ***	2.011 ***	0.461 ***	0.214 *	0.297 **	0.068
Shoot Ca2+	0.003 ns	19.815 ***	6.846 ***	3.024 ***	0.873 **	2.297 ***	0.978 ***	0.187
Root Ca2+	0.005 *	0.030 ***	0.007 ***	0.012 ***	0.003 **	0.011 ***	0.002 *	8.8 × 10−4
Shoot K+	88.888 ***	165.010 ***	440.657 ***	7.774 ns	5.620 ns	81.112 ***	35.561 ***	5.347
Root K+	42.013 ***	577.166 ***	483.782 ***	35.388 ***	4.986 *	13.310 ***	15.013 ***	1.562
Shoot P	0.010 ***	0.082 ***	0.007 ***	6.08 × 10−6 ns	0.001 ***	0.001 ***	2.38 × 10−4 ***	1.3–5
Root P	0.002 ***	0.003 ***	0.022 ***	4.68 × 10−5 *	3.6 × 10−4 ***	0.0014 ***	1.4 × 10−4 ***	1.0 × 10−5
Shoot As	64.723 ***	4805.875 ***	1365.578 ***	414.80 **	32.241 ***	105.58 ***	105.636 ***	2.391
Root As	4.869 ns	3643.594 ***	894.909 ***	125.17 ***	17.307 ***	110.45 ***	20.659 ***	1.704
Df	1	2	3	2	3	6	6	48

*, ** and *** = significant at 0.05, 0.01 and 0.001 levels, respectively; df = degrees of freedom; ns = non-significant; chl. a = chlorophyll a; chl. a/b ratio = chlorophyll a/b ratio; total chl. = total chlorophyll; A = net rate of photosynthesis; E = transpiration rate; g_s = stomatal conductance; Ψw = water potential; Ψs = osmotic potential; H₂O₂ = hydrogenperoxide; MDA = malondialdehyde; CAT = catalase; POD = peroxidase; SOD = superoxide dismutase; APX = ascorbate peroxidase; AsA = ascorbic acid; TSP = total soluble protein; RS = reducing sugars; NRS = non-reducing sugars; TPC = total phenolics content; TFC = total flavonoids content; TAC = total anthocyanin content; TFAA = total free amino acid.

). At 100 mg/kg of As-stress, Akbar and Pearl decreased shoot Ca²⁺ by 46.18%, 54.54%, respectively. Higher shoot Ca²⁺ was found in plants treated with thiamine and IAA pre-sowed seeds as combined and alone. The maximum shoot Ca²⁺ increase was recorded with the combined treatment of thiamine + IAA. At 100 mg/kg of As stress, shoot Ca²⁺ increased by 64.37%, 100% in Akbar and Pearl with combined treated seeds. Root Ca²⁺ decreased by 46.42, 19.62% in Akbar and Pearl, respectively, at 100 mg/kg As-stress. A maximum increase was observed in both varieties with thiamine + IAA as combined. Root Ca²⁺ increased by 133.33% and 32% in Akbar and Pearl at 100 mg/kg of As-stress with combined treated seeds. The highest As-stress level decreased shoot P (68.42, 62.5), root P (31.25, 38.88%), shoot K⁺ (32.97, 30%), root K⁺ (46.27, 44.95%) in Akbar and Pearl, respectively. A maximum increase in shoot and root K⁺ and P was observed with the combined treatment of thiamine and IAA under stress in both varieties. The thiamine and IAA combined application increased shoot P (44, 77%), root P (52.38, 178.18%), shoot K⁺ (76.19, 75.66%), root K⁺ (82.37, 131.57%), respectively, in Akbar and Pearl.

5.10. Uptake and Accumulation of As

A significant increase in shoot and root As contents was observed in both varieties under As-stress (Figure 5C,D). Maximum uptake was recorded under 100 mg/kg of As-stress in both varieties. As-stress levels increased As contents in the shoot and root hundreds times in Akbar and Pearl as compared to the control. The primed seeds with thiamine and IAA as alone and combined decreased the As contents in both varieties. As contents decreased by 39.34%, 46.49% in the shoot and root of Akbar, whereas Pearl depicted a decrease in As shoot and root by 66.79%, 58.36%, respectively, at 100 mg/kg of As-stress in combined treated seeds with thiamine and IAA.

6. Discussion

The As-stress constrained the maize plants to restrict growth (root and shoot length) and biomasses (root and shoot fresh and dry weights) to a significant extent by altering the biochemical mechanisms (Kaya et al., 2020 [34]; Khan et al., 2022 [35]). The stunted growth observed in both maize varieties was the result of chlorophyll set up disturbance, anatomical and biochemical changes. The agronomic characteristics (growth and biomass) of maize plants against As-stress represented different trends according to the variety of the different genetic pedigree (Atif and Perveen 2021 [55]). As a result of disrupted plant physiology under As-stress, the physio-biochemical nature of the plant was disturbed, ultimately leading plants to a remarkable decrease in growth that was already studied in previous research (Khan et al., 2022 [35], Bidi et al., 2021 [75]). The depiction of As tolerance against As-stress was already studied in our previous study (Atif and Perveen 2021 [55]) which correlated with chemical changes and the resistive nature of superior varieties as compared to sensitive ones. This is because of the presence of specific growth and development associated genes that expressed differentially under stress (Khare et al., 2022 [76]). The seed priming with IAA and thiamine increases the growth (shoot and root length) and biomasses (dry and fresh weight) of maize plants. Thiamine promotes the growth of plants under stress by improving photosynthetic contents, antioxidants and secondary metabolites (Jabeen et al., 2021 [41]. Correspondingly, endogenous enhanced levels of IAA stimulate the plant growth.

The decrease in chlorophyll pigments (Chl. a, b, Chl ratio a/b, total Chl. and carotenoids) under As-stress toxicity was prominent and was explored as the key indicator of heavy metal stress (Table 2 and Table 3). This is because As toxicity includes the photosynthetic degrading hormones instead of synthesizing hormones and compels plants to an oxidative burst (Zemanová et al., 2020 [77]; Ghorbani et al., 2020 [78]). The similar depression in chlorophyll attributes was concluded in a previous study by Tanveer et al. (2022) [79]. The seeds treated with thiamine as priming agents with thiamine and IAA were better in photosynthetic expression (Table 2 and Table 3) because thiamine as the priming agent can trigger the plant-defense system (Suohui et al., 2022 [80]). Hence, being as an essential nutrient, plant roots needed to synthesize it in sufficient amounts under harsh situations; however, exogenously uptake as seed priming could play a vital role in root growth and overall plant development (Goyer 2010 [81]). Thiamine is also known for combating or scavenging the ROS species, and hence, as a result of over ROS, it maintained the chlorophyll contents (Kaya et al., 2015 [42]). Accordingly, IAA also controlled the diminishing of chlorophyll pigments under As-stress (Table 2 and Table 3). This might be due to IAA as the main auxin that controlled the growth and development by stimulating the growth and having role in the process of photosynthesis and pigment formation. The photosynthetic activity increased with the priming of IAA, confirming the findings by Zhao et al. (2021) [82]. The novel insight of the current study was the combined priming application of both growth regulators which proved to be the most effective in the recovery of growth and photosynthesis characters.

The leaf gas exchange characters—rate of photosynthesis (A), transpiration rate (E), stomatal conductance (g_s)—of maize plants when exposed to As-stress showed a substantial decrease (Arikan et al., 2022 [18]). Different heavy metals including As decreased the gas exchange attributes (Anjum et al., 2017 [83]) that might be related to chlorophyll degradation and the disturbance of enzymes that take part in CO₂ fixation in the case of the rate of photosynthesis (Li et al., 2007 [84]). The other reason behind the decreased rate of E and A is due to low biomass production that increases the stomatal resistance (Šimonová et al., 2007 [85]). The improved gas exchange machinery was observed in plants that were primed with thiamine and IAA. Thiamine is the essential nutrient for plants and is recognized as a strong antioxidant agent against the plant stressed environment which in return improves the gas exchange characters.

As-stress also disturbs the plant-water relation and limits the growth (Vezza et al., 2018 [86]). Disturbance of plant water relation factors—leaf water potential (Ψ_w) and leaf osmotic potential (Ψ_s)—might be due to the altered structure of xylem both in roots and shoots. This might happen due to disturbed intercellular spaces, the water carriage capacity from root to shoot and the number of stomata under heavy metal stress (Rucińska-Sobkowiak 2016 [87]). The plant water relation attributes’ disturbance may be due to the result of the unbalanced water uptake through altered stomatal behavior (Singh et al., 2019 [88]). Imposed As-stress also has an obstacle for plants to regulate and retain the water in plant tissues (Anjum et al., 2017 [83]). Metal toxicity also creates an imbalance in the nutrient uptake that is associated with disturbed plant water relation (Muradoglu et al., 2017 [89]). The priming application with IAA and thiamine imparts better plant water relation in maize plants of both varieties. An explanation of greater leaf water and osmotic potential (Ψ_w and Ψ_s) by thiamine treatment might be due to the application of thiamine that lowered the osmotic stress by accumulating proline, and similar ameliorating effects of thiamine treatment on leaf water and proline contents have already been reported in maize (Sanjari et al., 2019 [90]) under Cd stress.

As-stressed maize plants exposed enhancement in enzymes as the result of over expressed ROS and imposed oxidative stress (Bhat et al., 2022 [25]; Mridha et al., 2022 [91]). Increases in APX SOD, POD in both maize varieties were in agreement with Suhel et al. (2022) [23], Atif and Perveen (2021) [55]. The SOD scavenges the ROS with direct participation against oxidative stress. Over-expression of SOD in plants induced tolerance against stresses. SOD converted the toxic O²⁻ radicals to molecular oxygen and H₂O₂ which later on was detoxified by GPX or CAT (Jung et al., 2019 [92]). Under stressed conditions, SOD regulated the intercellular ROS and physiological conditions (Faize et al., 2011 [93]). It showed amazingly unresponsive behavior in different plant species against stresses (Kliebenstein et al., 1998 [94]). In a similar way, POD breaks H₂O₂ at the cellular level with its different forms (Ros-Barcelo et al., 2002) [95]. Seeds primed with thiamine showed improved oxidative enzymes because thiamine limits the oxidative stress (Zhou et al. 2013 [96]). Thiamine acts as antioxidant after providing the NADH and NADPH (Asensi-Fabado and Munne-Bosch 2010 [97]). Correspondingly, IAA treated seed also showed minimal damages against antioxidant enzymes against As-stress Because it reduced heavy metals’ toxicity in roots which was ultimate factor for the lowering of damages (Bashri and Parasad 2015 [98]). Combined treated seeds with thiamine and IAA still provided the novel result of improving the antioxidant activities under As-stress.

The phenolic compounds including the flavonoids, AsA and anthocyanins are the secondary metabolites that play a key role in plant defense, and their activation depends on the intensity of stress. The current study depicted the decreased phenolic contents in both maize varieties under As-stress (Figure 3A) that was in agreement with Shamshir et al. (2022) [26] under As-stress. The phenolic compounds were also improved with the application of thiamine (Boubakri et al. 2013 [99]). AsA is necessary for ROS scavenging process and showed an increase against As-stress (Figure 3B). AsA has already been proved as the oxidative stress alleviator in the form of a priming agent (Elkelish et al., 2020 [100]). AsA accumulation enabled the plants to cope with oxidative stress with increased contents under stress in the current study (Figure 3B). Seeds primed with IAA improved AsA because IAA has the greater potential for improving the overall antioxidant capacity in oxidative stressed conditions (Madany et al., 2020 [101]). Anthocyanins are low molecular weight potent compounds that secure plants from heavy-metal-induced oxidative stress to a greater extent (Stambulska et al., 2018 [102]). The metal-induced increment in anthocyanin was already reported in Arabidopsis (Baek et al., 2012 [103]).

H₂O₂ and MDA are both considered as the stress indicators in oxidative stress and in terms of lipid peroxidation that reflects the plant strength to oxidative stress. When plants are facing As-toxicity, the oxidative potential was configured with increasing contents of both H₂O₂ and MDA (Bhat et al., 2022 [25]). The main reason was that the electron transfer during photosynthesis was inhibited, and both these biomolecules were found to be higher than the control, presenting oxidative stress under As-stress (Table 4) as confirmed by Kaya et al. (2022) [104] and Mishra et al. (2022) [29]. The cell membrane functions were disrupted with the over-accumulation of both these biomolecules (Zahra et al., 2018 [105]). The seed treated plants with thiamine and IAA showed decreases in both biomolecules (Table 4). Thiamine treated plants initiated the defensive machinery of plant and limited the concentrations of both by mitigating negative effects of As with reduced ROS which was also observed by Kaya et al. (2015) [42]. Sanjari et al. (2019) [90] found the role of thiamine to lower the H₂O₂ and MDA contents against oxidative damage by protecting the membranes. Thiamine application is useful in alleviating the toxic effects of stress and reduces H₂O₂ and MDA contents in different plants (Kaya et al. 2015 [42]). IAA reduces the heavy metal toxicity by improving membrane properties and decreasing the disorder of membrane organization (Bücker-Neto et al., 2017 [106]). However, the combined treatment of both growth regulators showed the better improvements (Table 4), while no study yet describes the combined exogenous application role on the biomolecules.

The current study indicated increased contents in osmolytes such as proline under As-stress (Dolui et al., 2022 [107]). The proline accumulation is being considered as an oxidative stress indicator along with sugars (Bidi et al., 2021 [75]). The TSS showed a definite increase under As-stress, while NRR and RS exhibited a decrease in their levels compared to the control (Table 5). The FAA and proline also were considered as the low molecular weight chemical compounds that accumulated in plants under stressed circumstances and coordinated with different plant regulators as a systemic strategy for metal detoxification in plants. Current results also demonstrated a marked increment in the deposition of proline and TFAA in maize plants growing in As-contaminated soil (Table 4 and Table 5). These results confirmed the scavenging ROS and antioxidative role of these molecules under oxidative stress and ROS accumulation. The influence of exogenously applied IAA and thiamine displayed a significant increase in endogenous levels of TFAA as alone and combined. The inflated accumulation of proline is correlated with better metal detoxification in plants (Hussain et al., 2019 [39]). IAA directly increased the protein and proline contents and counteracted with stress (Khalid and Aftab 2020 [108]). In contrast, RS and NRS decreased under stress, in agreement with Samanta et al. (2020) [109]. Current results showed that an As-induced increase in soluble sugars was substantially maintained in plants with thiamine and IAA individually and in combination. IAA decreased the protein contents under stress (Khalid and Aftab 2020 [108]). The mitigating effect of thiamine on maize cultivars might be due to its role as a coenzyme in various metabolic pathways such as sugar and protein metabolism (Goyer 2010 [81]). All attributes’ improvement through the exogenous application of thiamine and IAA as alone and combined was responsible to enhance the growth against As-stress in both maize varieties.

It is well documented that ionic changes reversibly trigger the antioxidant system. The initial set up that was disturbed in As-contaminated soil is a nutrient imbalance, and As travels from root to shoot and ultimately enters into edible parts of plants. The current study also concluded nutrient ions’ (K⁺, Ca²⁺, P) uptake disturbance when plants faced As toxicity (Figure 4A–D and Figure 5A–B). The translocation of different micro- and macronutrients was interrupted with As contamination which interferes negatively with the plant metabolism (Samanta et al., 2022 [110]). As-contamination significantly altered the nutrient ion uptake (Shamshir et al., 2022 [26]). Auxins promote stress tolerance and nutrient uptake by controlling the shoot/root growth ratio (Kurepa and Smalle 2022 [111]). In the present study, exogenously applied thiamine and IAA improved the nutrient uptake in maize plants under As stress and improved the availability of essential nutrients that competed with As. Rapala-Kozik et al. (2012) [112] suggested that thiamine mediates oxidative stress tolerance via salicylic acid and Ca²⁺-related signaling pathways.

As-translocation increased in plants from roots to shoots and in fruits (Kaya et al., 2020 [34]). As-contents were found more in roots than in shoots (Ruiz-Huerta et al., 2022 [113]). In sandy loam soils, the As-uptake in maize plants was observed more than in sand because the As translocation varies from soil to soil conditions and plants’ temporary conditions (Gulz et al., 2005 [114], Zheng et al., 2011 [115]). As ions were more pronounced in xylem sap as compared to roots in maize plants under As-stress that confirmed As accumulation in shoots (Su et al., 2010 [116]). The structural similarity of As with P facilitates As to accumulate in plants and travel to aerial parts of plants (Figure 5A,B). In the current findings, As contents were more in shoots which exhibits the uptake of As ions and accumulation in maize plants which was confirmed by (Kaya et al., 2020 [34]). Pearson correlation and principal component analysis are shown in Figure 6 and Figure 7.

7. Conclusions

The present investigations explored the differential expression with thiamine and Indole-3-acetic acid seed priming treatments of two maize varieties under Arsenic stress. The role of thiamine and IAA as individual and combined as seed priming proved to be better under oxidative stress by improving antioxidant enzymes, the plant water relation, the gas exchange machinery, lipid peroxidation and nutrient uptake capacity. However, the novel insight in this study was the combined application of thiamine and IAA as priming agents that was found as the best strategy of all to compete under As-induced alterations in plant biochemistry. The superiority of the variety was also studied as a response to As-stress resistance. Pearl proved itself better against captivated stressed levels. However, the study of the true potential of thiamine and indole-3-acetic acid involved in the complex molecular mechanisms associated with arsenic stress genes needs further research investigations.