Diphenyleneiodonium

Pretreatment of seeds with hydrogen peroxide improves deep-sowing tolerance of wheat seedlings

Askim Hediye Sekmen Cetinel a, Tolga Yalcinkaya a, Turgut Yigit Akyol b, Azime Gokce a,
Ismail Turkan a,*
a Department of Biology, Faculty of Science, Ege University, Bornova, 35100, Izmir, Turkey
b Department of Molecular Biology and Genetics, Aarhus University, 8000, Aarhus, Denmark

A R T I C L E I N F O

Abstract

Drought is a prevalent natural factor limiting crop production in arid regions across the world. To overcome this limitation, seeds are sown much deeper to boost germination by soil moisture produced by underground water. Seed pretreatment can effectively induce deep-sowing tolerance in plants. In the present study, we evaluated whether H2O2 pretreatment of seeds can initiate metabolic changes and lead to improved deep-sowing tolerance in wheat. Pretreatment with 0.05 μM H2O2 promoted first internode elongation by 13% in the deep-sowing tolerant wheat cultivar “Tir” and by 32% in the sensitive cultivar “Kıraç-66” under deep-sowing conditions, whereas internode elongation was inhibited by diphenyleneiodonium chloride. In contrast to Tir seedlings, H2O2 levels in the first internode of Kıraç-66 seedlings increased under deep-sowing condition in the H2O2-treated group compared to controls. Moreover, these seedlings had significantly lower catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX) activities but higher NADPH oxidase (NOX) and superoxide dismutase (SOD) activities under the same conditions, which consequently induced greater H2O2 accumulation. Contrary to Tir, both total glutathione and glutathione S-transferase (GST) activity decreased in Kıraç-66 after deep-sowing at 10 cm. However, H2O2 treatment increased the total glutathione amounts and the activities of glutathione-related enzymes (except GST and GPX) in the first internode of Kıraç-66. Taken together, these data support that H2O2 acts as a signaling molecule in the activation of antioxidant enzymes (specifically NOX, SOD, and CAT), regu- lation of both glutathione-related enzymes and total glutathione content, and upregulation of the cell wall- loosening protein gene TaEXPB23.

1. Introduction

Drought has destructive effects on both water resources and agri- culture. The severity of drought is expected to increase due to global warming via both decreased regional precipitation and increased evaporation (Dai, 2011; Senevirante et al., 2012). Arid areas are particularly prone to drought because their rainfall amount is critically dependent on a few rainfall events (Sun et al., 2006). Increments in drought severity, global aridity, and the global population will adversely affect food security (Gerland et al., 2014). Therefore, improvements in both crop production and agricultural sustainability will play a pivotal role in the lives of humankind. Since the manipulation of plant genetics through selective breeding is a valuable activity to achieve such im- provements, farmers have used tolerant species and varieties for a long time. For instance, grass varieties that have deep-sowing tolerance are cultivated in semi-arid regions. Generally, the cultivation of crop plants is achieved by sowing the seeds a few centimeters or less beneath the soil surface. However, in semi-arid areas, the upper layer of the soil is poorly moistened. As a result, shallowly sown seeds cannot absorb sufficient water for germination and the seedlings suffer from severe drought stress. To overcome this limitation, seeds are sown much deeper to promote germination with the help of soil moistened by underground water. Deep-sowing can also assist in reducing devastation of seeds by birds and rodents (Brown et al., 2003) and in avoiding phytotoxicity associated with some pre-emergent herbicides (O’Sullivan et al., 1985). Nevertheless, this method can only be achieved with the use of deep-sowing tolerant cultivars whose seedlings elongate sufficiently to allow emergence from extreme soil depths. For example, Hopi maize, a local corn variety able to emerge from depths of 25–30 cm by mesocotyl
elongation, is cultivated in the southwestern region of the United States and the western region of Mexico (Collins, 1914; Troyer, 1997). Another example of a deep-sowing tolerant cultivar is a local variety of wheat called “Hong Mang Mai” (Takeda and Takahashi, 1999), whose seed- lings emerge from a soil depth of 18–20 cm via first internode elongation (Suge et al., 1997). Similarly, “Tir” is a deep-sowing tolerant variety of wheat cultivated in the Eastern Anatolian region of Turkey (Yagmur and Kaydan, 2009). Its seeds can be sown up to 20 cm deep in the soil and its seedlings can emerge from this depth by elongating their first internode. As shown by these examples of various plant species, elongation of plant organs such as the first internode, mesocotyl, or both can maintain a shoot apical meristem elevated above the soil surface to enable deep-sowing tolerance (McCall, 1934; Hoshikawa, 1969). There are only a few studies in the literature that contribute to the understanding of the deep-sowing tolerance mechanism in wheat, which is basically explained by first internode elongation and a hormone response. Several endogenous factors, such as hormones (e.g., gibberellin and ethylene) and minerals (potassium), are reported to be involved in first internode elongation (Suge et al., 1997; Chen et al. 2001, 2003; Nishizawa et al., 2002).
The dual roles of reactive oxygen species (ROS) in plant biology were highlighted by Mittler et al. (2004): on one hand, ROS act as important signal transduction molecules during stress response, plant growth, and development, on the other hand, ROS are toxic byproducts of aerobic metabolism that accumulate in cells during different stress conditions. As a ROS, H2O2 has been studied in recent years as a signaling molecule in plant growth and development processes such as tip growth of root hairs (Chen et al., 2003; Achard et al., 2008), root gravitropism (Jiang et al., 2012), lateral (Su et al., 2006) and adventitious root (Li et al., 2009) development, coleoptile elongation (Schopfer, 1996), pollen tube growth (Potocký et al., 2007), and cell wall development (Potikha et al., 1999). However, reports on the effects of H2O2 on plant growth and development are limited.

Pre-sowing treatments and priming of seeds are effective strategies to overcome environmental stress (Ashraf and Foolad, 2005). Priming seed strategies such as osmopriming, hormonal priming, and hydropriming involve treatments with osmotica, hormones, or water, respectively, to induce pre-germination metabolic changes. These changes usually have profound effects on both the germination rate and uniformity of seedling emergence, especially under stressful conditions (Ashraf and Foolad, 2005). However, reports on the use of stress signaling agents such as H2O2 are scarce.

Pretreatment of pea seeds with H2O2 has been reported to increase germination rate and promote seedling growth (Barba-Espin et al., 2010). Li et al. (2007, 2009) reported that exogenous H2O2 promoted the formation and development of adventitious roots in both mung bean and cucumber seedlings. However, Jiang et al. (2012) found that exogenous H2O2 inhibited pea root gravitropism. Schopfer (1996) showed that an excess amount of exogenous H2O2 inhibited coleoptile growth and decreased cell wall extensibility in maize. As can be seen, most previous studies have focused on the effects of H2O2 pretreatment on the growth and development of seeds, root, seedlings, and coleoptile. By contrast, the role and mechanism(s) of H2O2 pretreatment on the induction of first internode elongation, as a strategy to overcome deep-sowing germination failure, remain unclear.

To fill this knowledge gap, the present study examined the role of H2O2 on elongation growth in two Triticum aestivum cultivars differing in deep-sowing tolerance, namely the deep-sowing tolerant “Tir” and the deep-sowing sensitive “Kıraç-66”. The interactions of H2O2 with the activities of antioxidant and glutathione-related enzymes during first internode elongation were also examined. To do so, for the first inter- node of Tir and Kıraç-66 sown 10 cm beneath the soil surface, we determined: (1) the effects of H2O2, deep-sowing (DS), or their combi- nation on the length of the first internode; (2) the interactions between H2O2 and changes in the NADPH oxidase (NOX) activity, RBOHD and RBOHF gene expression, antioxidant enzyme activities [superoxide dismutase (SOD) and its isoenzymes, catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX)], total glutathione content, and glutathione-related enzyme activity [glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST), and its isoenzymes] in the first internode and (3) the effects of H2O2 on the expression of genes related to cell wall loosening/stiffening, such as those coding for extensin-like (TaExtLP) and expansin (TaEXPB23) proteins.

2. Materials and methods
2.1. Plant materials and treatments

In the present work, the wheat cultivars Triticum aestivum L. Tir and Kıraç-66 were used. In contrast to DS tolerant Tir, Kıraç-66, which is cultivated in the Central Anatolian region, is classified as a moderately drought-resistant wheat cultivar (Ozturk and Aydın, 2017) and is known to be sensitive to DS. Tir and Kıraç-66 seeds were obtained from Prof. Dr.

Muzaffer Tosun of Ege University, Faculty of Agriculture, Bornova, I˙zmir, Turkey and the Anatolia Agricultural Research Institute, Eski-
s¸ehir, Turkey, respectively. Seeds were selected for uniformity before use. Uniform-sized seeds were sterilized by treatment with 70% ethanol for 1 min followed by 5% sodium hypochlorite for 3 min. After sterili- zation, seeds were rinsed with distilled water three times. Preliminary studies using various concentrations of H2O2 (0, 0.1, 0.25, 0.5, 1, 1000, 40000 μM) have shown that we did not observe much difference be- tween 0.1 μM and 0.25 μM. Therefore, seeds (100 seeds for each repli- cate) were transferred to 0, 0.05 μM, 0.5 μM, 1 μM, 1000 μM or 40000 μM H2O2 solution and immersed for 24 h in the dark. The seeds, which were treated with either deionized water (dIH2O) or different H2O2 concentrations, were then sown on the surface of 2-cm thick 1% agar blocks (200 ml) placed at the bottom of pots (12 cm in diameter, 20 cm in length) and covered with either 2 cm (shallow-sowing, Ctrl) or 10 cm (DS) of soil [peat/vermiculite/perlite (7/2/1, v/v/v)]. The agar blocks placed in the pots served as a water reserve to allow seed germination. To eliminate the effects of ROS produced by NADPH oxidase, 10 μM diphenyleneiodonium chloride (DPI), an NADPH oxidase inhibitor, was added to dIH2O in which the seeds had been immersed in. Plants grown from seeds imbibed with H2O2, DPI, or dIH2O were grown in darkness in a JSPC-420C plant growth chamber (JSR, Gongju City, Republic of Korea) at 25 ◦C. After 10 days, the first internode elongation of the wheat seedlings was measured.
The experimental treatments were as follows:
(1) Control (Ctrl), Tir and Kıraç-66 seedlings whose seeds were sown 2 cm deep;
(2) H2O2 alone, Tir and Kıraç-66 seedlings whose seeds were immersed in dIH2O containing 0.05 μM H2O2 for 24 h in the dark before being sown 2 cm deep;
(3) DS alone, Tir and Kıraç-66 seedlings whose seeds were sown 10 cm deep;
(4) Co-application of H2O2 with DS (DS + H2O2), Tir and Kıraç-66
seedlings whose seeds were immersed in dIH2O containing 0.05
μM H2O2 for 24 h in the dark before being sown 10 cm deep; and
(5) Co-application of DPI with DS (DS + DPI), Tir and Kıraç-66 seedlings whose seeds were immersed in dIH2O containing 10 μM DPI for 24 h in the dark before being sown 10 cm deep.

2.2. Determination of growth parameters

The length of the first internodes of the DS tolerant (Tir) and sensi- tive (Kıraç-66) wheat cultivars whose seeds were sown at different depths (2 or 10 cm) were measured using a measuring scale.

2.3. Determination of H2O2 content and lipid peroxidation

H2O2 production in the first internode was assessed using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, Molecular Probes, UK) according to the manufacturer’s instructions. The first in- ternodes were ground in liquid nitrogen, followed by the addition of 3 ml phosphate buffer (20 mM K2HPO4, pH 6.5) to 1 g ground frozen tissue. A 50 μl sample extract was incubated with 10 U ml—1 horseradish peroxidase and 100 mM Amplex Red reagent (10-acetyl-3, 7-dihydro- phenoxazine) at room temperature for 30 min in the dark. Fluores- cence was measured using a Microplate Spectrofluorometer (Thermo/ Varioskan Flash) at excitation/emission of 530/590 nm. The experi- ments were repeated at least four times independently in duplicates. Lipid peroxidation [reflected by the thiobarbituric acid reactive substance (TBARS) content] was measured by grinding leaf tissue (200 mg) into a fine powder in liquid nitrogen. The tissue was homogenized in 800 μl cold 5% (w/v) trichloroacetic acid. The homogenate was centrifuged at 12 000×g for 30 min and further process based on the method described by Madhava Rao and Sresty (2000). The concentra- tion of TBARS was calculated using an extinction coefficient of 155 mM cm—1. All tests were performed in triplicates.

2.4. Determination of enzyme activity

Enzyme extraction was performed at 4 ◦C to minimize protease ac- tivity. Subsequently, 0.1 g sample was homogenized in 300 μl 50 mM
Tris-HCl pH 7.8 containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (v/v) Triton™ X-100, 1 mM phenylmethylsulfonyl fluo- ride (PMSF), and 2% (w/v) polyvinylpolypyrrolidone (PVPP). For the APX activity assay, 5 mM of ascorbate (AsA) was added to the extraction buffer. Samples were centrifuged at 14000×g for 10 min at 4 ◦C. The supernatants were then collected and used for determining protein
content and enzyme activities. The total soluble protein content of the enzyme extracts was ascertained according to Bradford’s method (1976) using bovine serum albumin as a standard. All spectrophotometric an- alyses were performed using a Shimadzu (UV 1600) spectrophotometer.NADPH oxidase (NOX, EC 1.6.3.1) activity was evaluated according to the method described by Jiang and Zhang (2002). The reaction mixture contained 50 mM Tris-HCl buffer pH 7.5, 0.5 mM sodium 3, 3′-(—[(phenylamino)carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), 100 μM NADPH⋅Na4, and 10 μl sample. After the addition of NADPH, the XTT reduction was screened at 470 nm. The corrections of background production were determined in the presence of 50 U SOD. Activity was calculated using an extinction coefficient of 2.16 × 104 M—1 cm—1. One unit of NOX was defined as 1 nmol ml—1 XTT oxidized min—1.

SOD (EC 1.15.1.1) activity was assayed by its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT) at 560 nm (Beauchamp and Fridovich, 1971). One unit of SOD was defined as the amount of enzyme that inhibited 50% NBT photoreduction.
POX (EC1.11.1.7) activity assay was performed according to Herzog and Fahimi (1973). The reaction mixture contained 3,3′-dia- minobenzidine-tetra hydrochloride dihydrate solution containing 0.1% (w/v) gelatine and 150 mM sodium phosphate citrate buffer pH 4.4 and 0.6% H2O2. The increase in absorbance at 465 nm was screened for 3 min. A unit of POX activity was defined as mmol H2O2 decomposed ml—1 min—1. CAT (EC 1.11.1.6) activity was estimated according to the method of Bergmeyer (1970), which measures the initial rate of H2O2 decompo- sition at 240 nm (molar extinction coefficient of 43.6 M—1 cm—1). The decrease in absorption was screened for 1 min and 1 μmol H2O2 min—1 was defined as 1 unit of CAT. APX (EC 1.11.1.11) activity assay was performed according to the method of Nakano and Asada (1981). This analysis depends on the decrease in absorbance at 290 nm as ascorbate is oxidized. The reaction mixture contained 50 mM potassium phosphate buffer pH 7.0, 0.5 mM ascorbate, 0.1 mM EDTA Na2, 0.1 mM H2O2, and 0.1 ml enzyme extract in a final volume of 1 ml. The concentration of oxidized ascorbate was calculated using an extinction coefficient of 2.8 mM—1 cm—1. One unit of APX was defined as 1 mmol ml—1 ascorbate oxidized min—1. GR (EC 1.6.4.2) activity was measured according to Foyer and Halliwell (1976). NADPH oxidation was screened at 340 nm. Activity was calculated using the extinction coefficient of NADPH (6.2 mM—1 cm—1). One unit of GR was defined as 1 μmol GSSG reduced min—1.

DHAR (EC 1.8.5.1) activity was assayed according to the method of Nakano and Asada (1981). This analysis depends on the increase in absorbance at 265 nm for 1 min. The reaction mixture contained 2.5 mM GSH, 0.2 mM dehydroascorbate (DHA), and 0.1 mM ethyl- enediaminetetraacetic acid tetrasodium salt dihydrate (EDTA) in 50 mM K-phosphate (pH 7.0). One unit of DHAR was defined as nmol DHA recycled ml—1 min—1. GST (EC 2.5.1.13) activity was assayed according to the method of Habig et al. (1974). This analysis depends on the decrease in absorbance at 265 nm for 1 min. The reaction mixture contained 0.1 M Na-phosphate (pH 6.5), 1 mM GSH, 1 mM GSH, and 1 mM CDNB. One unit of DHAR was defined as nmol DHA recycled ml—1 min—1. GPX activity was measured according to Li and Yi (2012). This analysis depends on the decrease in absorbance at 340 nm for 1 min. The reaction mixture contained 0.2 mM NaDPH, 1 mM sodium azide, 1 mM
GSH, 1 U GR, and 2 mM H2O2. One unit of GPX was defined as the amount of enzyme catalyzing the oxidation of 1 mmol of NADPH min—1.
The specific enzyme activity for all enzymes is expressed in unit mg—1 protein.

2.5. Identification of SOD and GST isoenzymes

For the separation of SOD isoenzymes, samples containing 50 μg protein per well were subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) in 4.5% stacking and 12.5% separating gels under a constant current (70 mA) at 4 ◦C. SOD activity was detected by photochemical staining with riboflavin and NBT according to the method of Beauchamp and Fridovich (1971). The unit activity of each SOD isoenzyme was calculated by running an SOD standard from bovine liver. SOD isoenzymes were identified by incu- bating gels with SOD inhibitors before staining, namely 2 mM KCN (to inhibit Cu/Zn-SOD activity) and 3 mM H2O2 (to inhibit Cu/Zn-SOD and Fe-SOD activities) according to Vitoria et al. (2001) (Mn-SOD activity is resistant to both inhibitor treatments).

For the separation of GST isoenzymes, samples containing 50 μg protein per well were subjected to nondenaturing PAGE (Laemmli, 1970) in 4.5% stacking and 10% separating gels under a constant cur- rent (70 mA) at 4 ◦C. After separating the proteins, gels were incubated in 0.1 M K–P buffer (pH 6.5) for 10 min. These gels were subsequently transferred to a dye solution containing 4.5 mM GSH, 1 mM CDNB, and 1 mM NBT in 0.1 M K–P buffer (pH 6.5).All gels were photographed with a Vilber Lourmat gel imaging sys- tem and analyzed using the Bio-Capt software package (Vilber Lourmat, Marne la Vall´ee, France). For the isoenzymes, induction was quantified as a percentage (%) by recording the density of each band.

2.6. Determination of glutathione (GSH) and oxidized glutathione (GSSG) contents

The GSH and GSSG contents were determined according to the method of Queval and Noctor (2007). Extraction was performed at 4 ◦C. Leaf tissue (0.1 g) was ground in liquid nitrogen and extracted with 1 ml 0.2 M HCl. Subsequently, samples were centrifuged at 16 000×g for 10 min. The supernatant (0.5 ml) was neutralized with approximately 0.4 ml 0.2 M NaOH in the presence of 50 μl 0.2 M NaH2PO4 (pH 5.6). The pH of the neutralized acid extracts was between 5 and 6. GSH content was determined using an enzyme cycling assay and screening the change in absorbance at 340 nm, whereas GSSG content was determined using 2-vinylpyridine derivatization followed by an enzyme cycling assay.

2.7. RNA isolation and cDNA synthesis

Total RNA was isolated from 0.1 g plant tissue (first internode) using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. To remove residual genomic DNA, the total RNA samples were treated with DNase I (Fermentas). Extracted high- purity total RNA from first internode tissues was quantified using NanoDrop (Thermo NanoDrop). Subsequently, the integrity of the RNA samples was controlled by running on 1% formaldehyde agarose gel and first-strand cDNA synthesis was performed following the manufacturer’s instructions (Roche High Fidelity cDNA synthesis kit). These cDNAs were used as templates for quantitative reverse transcription polymerase chain reaction (qRT-PCR). The amount of RNA in each reaction was normalized to the T. aestivum β-actin gene.

2.8. Gene expression analyses by qRT-PCR

Three independent experiments were performed for qRT-PCR assays using the StepOne Plus™ Real-Time PCR System (Applied Biosystems). Power SYBR Green Master Mix was used (Applied Biosystems) for these analyses. The qRT-PCR reactions contained 75–200 ng cDNA as a tem- plate, 10 pmol primers (for each forward and reverse primers), and 12.5 μl Power SYBR Green Master Mix in a total volume of 25 μl. The con- ditions for PCR amplification were as follows: For TaRBOHD and TaR- BOHF, 95 ◦C for 5 min, 50 cycles at 94 ◦C for 15 s, 60 ◦C for 15 s, and
72 ◦C for 10 s. For β-actin and extensin-like (TaExtLP): 95 ◦C for 5 min, 45 cycles at 95 ◦C for 30 s, and 55 ◦C for 1 min. For expansin (TaEXPB23): 95 ◦C for 5 min, 40 cycles at 94 ◦C for 20 s, 60 ◦C for 30 s, and 68 ◦C for 35 s. Data analyses were performed using StepOne Plus software according to Livak and Schmittgen (2001). Accordingly, each sample was examined in triplicates using relative quantification analyses. This method normalizes the expression of specific gene versus the control reference with the formula 2-ΔΔCT, where 2-ΔΔCT = ΔCT-arbitrary constant, ΔCT = CT specific gene — CT reference gene. The primers and the amplicon sizes for the housekeeping gene β-actin (AY663392, which was used as a control reference), TaRBOHD (AK335454), TaRBOHF (AY561153), extensin-like (TaExtLP), and expansin (TaEXPB23, RT23) are given in Table 1. All primers were synthesized by Sentromer DNA Technologies (Istanbul, Turkey).

2.9. Statistical analysis

The experiments were repeated twice, and three biological replicates were used from each experiment for all analyses (n = 6). Results are expressed as the mean ± standard error of the mean. Groups were compared using Student’s t-tests. In the figures, different letters above the bars indicate significant differences between the control and treat- ment groups at the P ≤ 0.05 level according to the least significant difference (LSD) test.

3. Results
3.1. First internode length

H2O2 is known to have either stiffening or softening effect on the cell wall, depending on its content level in the cell. To reveal the role of H2O2 as a seed pretreatment, DS tolerant Tir seeds were treated with increasing H2O2 concentrations (0.05 μM, 0.5 μM, 1 μM, 1000 μM or 40000 μM) for 24 h before sowing 10 cm beneath the soil surface (Fig. 1).

Fig. 1. First internode length of seedlings arising from deep-sowing tolerant T. aestivum Tir seeds treated with increasing H2O2 concentrations (0.05 μM, 0.5 μM, 1 μM, 1000 μM or 40000 μM) under deep-sowing conditions (10 cm beneath the soil surface). Data show the mean first internode length values of 70 plants (for each concentration) and the standard error (vertical bars).

Our results showed that pretreatment with 0.05 μM H2O2 triggered a significant increase in the growth of the first internode of Tir seedling. However, higher H2O2 concentrations inhibited its growth. Therefore, activities of the antioxidant- and glutathione-related enzymes in the first internode of both DS tolerant and sensitive wheat seedlings grown from seeds imbibed with 0.05 μM H2O2 and sown at two depths were compared. The first internode length increased by 12% in Tir, but did not change in Kıraç-66, in DS conditions. Pre-sowing seed treatment with 0.05 μM H2O2 triggered a significant increase in the first internode elongation of 32% in Kıraç-66, but only 13% in Tir, in DS conditions. On the other hand, in shallow-sowing conditions, no significant effect of H2O2 on first internode elongation was observed for either cultivar (Fig. 2).

To examine the effect of H2O2 production by NADPH oxidase (NOX) on first internode elongation of both Tir and Kıraç-66 cultivars under DS conditions, inhibition assays with DPI were conducted on their seeds. First internode elongation of both cultivars was significantly inhibited by 10 μM DPI under DS conditions. DPI inhibited elongation of the first internode by 21% in Tir, but only by 13% in Kıraç-66, compared to the control group. These results suggested that first internode elongation was more sensitive to NOX-mediated H2O2 production at DS conditions than under shallow-sowing conditions (Fig. 2).

3.2. First internode elongation ability

To determine the first internode elongation abilities of the deep- sowing tolerant (Tir) and sensitive (Kıraç-66) wheat cultivars, the length of the first internode of 70 wheat seedlings germinated under DS conditions was measured. The length distribution of the first internode of both Tir and Kıraç-66 seedlings is shown in Fig. 3.

As shown in Fig. 3A, the first internode length differed significantly between the two cultivars. A large number of Kıraç-66 seedlings had a first internode length ranging between 4 and 6 cm, but very few seed- lings had internodes longer than 7 cm. By contrast, the Tir seedlings had longer internodes in the range of 7–12 cm under DS conditions.Fig. 3B shows the distribution of the first internode length of both Tir and Kıraç-66 seedlings arising from seeds imbibed in 0.05 μM H2O2.

H2O2 treatment caused a significant two-fold increase in the number of Tir plants with first internodes between 10 and 12 cm long at the 10-cm sowing depth. Moreover, the number of the Kıraç-66 seedlings with a first internode length between 7 and 9 cm under DS conditions increased four-fold in the presence of H2O2.In the presence of DPI, the length of the first internode was less than 10 mm in about one-third of the Kıraç-66 seedlings under DS conditions (Fig. 3C). Only 29 Kıraç-66 seedlings had a first internode that was 40–60 mm long. Similarly, the number of Tir seedlings with a first internode length between 10 and 12 cm also decreased (1.2-fold) by DPI treatment.

3.3. First internode elongation rate

Fig. 4 shows the time-course assessment of the first internode growth of both Tir and Kıraç-66 seedlings under DS conditions arising from seeds in the presence (+) or absence (—) of 0.05 μM H2O2.

Fig. 2. Photographs of 10-day-old Kıraç-66 and Tir seedlings treated with either H2O2 (+H2O2) or diphenyliodo- nium chloride (+DPI) under shallow-sowing (2 cm, Ctrl) or deep-sowing (10 cm, DS) conditions. Arrowheads indicate first node. Scale bar = 10 cm.Mean values ± standard error (n = 20–25) are presented. (A) Effects of DS, 0.05 μM H2O2, and 10 μM DPI on the first internode elongation of deep- sowing tolerant (Tir) and sensitive (Kıraç-66) wheat cultivars at different sowing depths [2 cm (Ctrl, shallow- sowing) and 10 cm (DS)]. (B) For the DPI treatment, Tir seeds were immersed in either 10 or 20 μM DPI before being planted on the surface of agar blocks. Since 20 μM DPI caused a significant decrease in the germination of Tir seeds, we did not obtain a sufficient sample for measuring the H2O2 content. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the least significant difference test. Fig. 3. (A) First internode length of 70 seedlings arising from DS tolerant T. aestivum Tir and sensitive Kıraç-66 wheat seeds under only DS (10 cm) conditions according to the elongation ability test. (B) First internode length of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with 0.05 μM H2O2 under DS (10 cm) conditions according to the elongation ability test. (C) First internode lengths of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with 10 μM diphenyliodonium chloride under DS (10 cm) conditions according to the elongation ability test. Fig. 4. Time-course assessment of the elongation of the first internode of wheat seedlings affected by H2O2 pretreatment. Plots show the means and standard errors (vertical bars) of the first internode lengths of 70 plants. The first internodes of Tir seedlings rapidly elongated from days 6–10, whereas those of Kıraç-66 seedlings only elongated from days 6–8 (Fig. 4A and B). Elongation rates were particularly remarkable from days 6–8 for both cultivars. Compared with controls (Ctrl), DS treatment caused a significant increase in the elongation rate of the first internode of Tir seedlings between days 6 and 8, regardless of H2O2 treatment (Fig. 4A). In contrast, DS did not change the elongation rate of the first internode from days 8–10. However, under DS conditions, soaking seeds with 0.05 μM H2O2 (D + H) increased the elongation rate by 37%.Therefore, the highest elongation rate in the first internode was recorded in seedlings germinated from seeds imbibed in 0.05 μM H2O2 under DS conditions (Fig. 2B and C). 3.4. Endogenous H2O2 content Soaking seeds in 0.05 μM H2O2 increased the endogenous H2O2 content in the first internode of the Kıraç-66 seedlings by 15%, but decreased it by 18% in Tir seedlings, under DS conditions (Fig. 5). DPI treatment caused a significant decrease (35%) in the endogenous H2O2 content in the first internode of Tir seedlings under DS treatment. Similarly, the endogenous H2O2 content recorded for Kıraç-66 decreased after DPI treatment, but this decrease was not statistically significant. 3.5. NADPH oxidase activity NOX activity was enhanced 2.3-fold in the first internode of the deep- sowing tolerant Tir cultivar, but reduced in the Kıraç-66 cultivar, under DS treatment. H2O2 pretreatment triggered a significant increase in NOX activity in the first internode of both cultivars under the same condi- tions. However, the application of 10 μM DPI under DS conditions prevented the increase in NOX activity in the first internode of Tir and Kıraç-66 by 27% and 15%, respectively (Fig. 6A). On the other hand, under shallow-sowing (Ctrl) conditions, H2O2 pretreatment increased NOX activity in the first internode of Tir seedlings, but not in the first internode of Kıraç-66 seedlings. Fig. 5. H2O2 content of the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or DPI at 2 sowing depths ([2 cm, control, Ctrl] and [10 cm, deep-sowing, DS]). H2O2 and DS +H2O2: wheat seedlings germinated from seeds imbibed in 0.05 μM H2O2 before sowing at 2 cm or 10 cm beneath the soil surface, respectively. DS + DPI (co-applica- tion of DPI with deep-sowing): seedlings obtained from seeds imbibed for 24 h in dIH2O, containing 10 μM DPI before sowing at 10 cm deep. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the LSD test. To better understand the effect of pretreatment with H2O2 on NOX/ RBOH-mediated ROS signaling, the expression patterns of RBOHD and RBOHF were determined in the first internode of both wheat cultivars by qRT-PCR (Fig. 6B and C). According to the expression values, RBOHD was upregulated (2.39-fold) in Tir seedlings under DS conditions. In contrast, RBOHF expression was not responsive to DS. However, in Kıraç-66, the expression of RBOHF decreased under DS conditions, while the expression of RBOHD was slightly upregulated (1.31-fold). H2O2 pretreatment increased (2.8-fold) RBOHD expression in the first internode of both cultivars’ seedlings under shallow-sowing (Ctrl) conditions. However, the expression of RBOHF was not significantly changed. Under deep-sowing conditions, H2O2 pretreatment did not change the expressions of these genes in the first internode of Tir seedlings (DS + H2O2), but increased (1.8-fold) the expression of RBOHD in Kıraç-66 seedlings. However, the expression of RBOHF was not induced by H2O2 pretreatment in this cultivar. By contrast, both RBOHD and RBOHF genes were downregulated by DPI treatment in the first internode of both cultivars under DS conditions. 3.6. Activities of H2O2-producing/-metabolizing enzymes in the first internode To investigate whether H2O2 pretreatment caused the induction of H2O2-metabolizing enzymes in the first internode of both Tir and Kıraç- 66 seedlings germinated under DS conditions, the activities of antioxi- dant enzymes, such as SOD, CAT, POX, and APX, were measured. SOD activity recorded in the first internode was much greater in Tir seedlings than in Kıraç-66 seedlings at both sowing depths (Fig. 7A). SOD activity was not changed by H2O2 pretreatment under shallow- sowing conditions in either cultivar. However, SOD activity increased by 32% in the first internode of Tir seedlings at the 10 cm sowing depth, while H2O2 pretreatment did not change SOD activity under the same conditions. DS did not result in any increase in SOD activity in the first internode of Kıraç-66 seedlings, although imbibition of seeds with 0.05 μM H2O2 increased SOD activity by 16%. DPI treatment decreased SOD activity by 1.72-fold in the first internode of Tir seedlings under DS conditions. However, SOD activity in Kıraç-66 seedlings was not significantly decreased by DPI.Two SOD isoenzymes, namely Cu/Zn-SOD and Mn-SOD, were iden- tified in the first internode of both cultivars (Fig. 7B). The highest SOD activity was observed in the first internode of Tir seedlings under DS treatment (DS and DS + H2O2) due to the high activities of both Cu/Zn- SOD and Mn-SOD. However, DPI decreased the activities of these isoenzymes. The activities of Cu/Zn-SOD and Mn-SOD did not change in the first internode of Kıraç-66 seedlings under DS conditions. However, similar to Tir, H2O2 pretreatment resulted in a significant increase in the activities of these isoenzymes under the same conditions (DS + H2O2) in Kıraç-66 seedlings. H2O2 pretreatment increased the activities of both CAT and POX enzymes, but did not change APX activity, in the first internode of the seedlings of both cultivars under shallow-sowing conditions (Fig. 8A–C). Under DS treatment, the activities of both CAT and POX differed significantly from that of APX. In Tir, CAT and POX activities signifi- cantly decreased by 61% and 25%, respectively, but APX activity did not change under DS conditions. In Kıraç-66, CAT and POX activities increased by 270% and 45%, respectively, while APX activity did not change. On the other hand, H2O2 pretreatment resulted in a 53% decrease in CAT activity, but increased (17%) POX activity, under DS conditions. However, this treatment did not change APX activity in the first internode of Tir seedlings under DS conditions. Similarly, CAT ac- tivity in Kıraç-66 seedlings decreased after H2O2 pretreatment. In contrast to Tir, H2O2 pretreatment of Kıraç-66 seeds resulted in decreased POX activity but increased APX activity in the first internode of seedlings germinated under DS conditions. DPI treatment inhibited the activities of CAT, POX, and APX in the first internode of Tir seedlings under DS conditions. For Kıraç-66 seedlings, CAT and POX activities decreased by 2.56-fold and 10%, respectively, after DPI treatment. However, APX activity did not change. 3.7. Redox forms of glutathione in the first internode To investigate the effect of H2O2 on redox forms of glutathione during first internode elongation, reduced GSH, GSSG, and total GSH contents were determined in the first internode of Tir and Kıraç-66 cultivars (Fig. 9). The GSH content increased in the first internode of both cultivars under DS conditions. The greatest increase (78%) was observed in the first internode of the Tir seedlings. DS resulted in a significant increase in the GSSG content in the first internode of Tir seedlings, but not in that of Kıraç-66 seedlings where it decreased by 59%. H2O2 pretreatment resulted in a greater increase in the GSH con- tent in the first internode of Tir seedlings than of Kıraç-66 seedlings under DS conditions. In contrast to GSH, GSSG content was not affected by H2O2 treatment in Tir, but increased two-fold in Kıraç-66 under DS conditions. Under the same conditions, the total glutathione content in Kıraç-66 seedlings was not significantly changed, but it increased 1.8- fold in Tir seedlings. The highest total glutathione content was observed in the first internode of H2O2-treated Tir seedlings germinated under DS conditions, specifically due to the increased GSH amounts. DPI treatment resulted in decreased GSH and GSSG contents in the first internode of both cultivars under both deep- and shallow-sowing conditions. Fig. 6. (A) NADPH oxidase (NOX) activity in the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or diphenyleneiodonium chloride at 2 sowing depths [(2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)]. Different letters above the bars indicate sig- nificant differences among treatments at the P < 0.05 level, according to the least significant difference test. (B, C) quantitative reverse transcription polymerase chain re- action results of RBOHD and RBOHF expression, respectively, in the first inter- node of seedlings. Groups that are significantly different from the control, according to Student’s t-tests, are indicated by *, P < 0.05. 3.8. Activities of glutathione-related enzymes in the first internode To investigate the role of H2O2 seed treatment on the activities of glutathione-related enzymes, GR, DHAR (dehydroascorbate reductase), GPX (glutathione peroxidase), and GST activities were determined in the first internode of both Tir and Kıraç-66 cultivars under deep- and shallow-sowing conditions (Fig. 10A–C).Soaking seeds with 0.05 μM H2O2 increased GR activity by 57% and 44% in the first internodes of Tir and Kıraç-66 seedlings, respectively, under shallow-sowing treatment compared to controls (Fig. 10A). Similarly, GR activity also increased under DS conditions, with the highest increase observed for the first internode of Tir seedlings. Under DS conditions, GR activity was amplified in the first internode of the H2O2-pretreated groups by 17% and 27% in Tir and Kıraç-66 cultivars, respectively. On the other hand, DPI caused a significant decrease in GR activity in both cultivars. Fig. 7. (A) Total activity of superoxide dismutase (SOD) and (B) its isoenzyme patterns in the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with H2O2 or diphenyleneiodonium chloride at 2 sowing depths [(2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)]. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the least significant difference test. DHAR activity, recorded in the first internode of both cultivars, was induced by H2O2 pretreatment under shallow-sowing conditions compared with controls (Fig. 10B) and was also increased by DS. The highest DHAR activity was observed in Tir seedlings germinated under DS conditions. H2O2 pretreatment did not change the increase in DS induced GR activity recorded in Tir seedlings, but decreased GR activity in Kıraç-66 seedlings. DPI initiated a significant decrease in DHAR ac- tivity in both cultivars under DS conditions. H2O2 pretreatment increased GPX activity by 37.8% in the first internode of Tir seedlings under DS conditions, but did not change GPX activity in Kıraç-66 seedlings, compared with the control group (Fig. 10C). Moreover, DS did not result in any change in GPX activity in either cultivar. However, H2O2 pretreatment increased GPX activity by 23% in Tir seedlings, but reduced it by 20% in Kıraç-66 seedlings, under DS conditions. DPI decreased GPX activity in both cultivars. DS resulted in a significant increase in GST activity in Tir seedlings due to increased activities of GST1, GST3, and GST4 isoenzymes (Fig. 11A and B) compared to controls. H2O2 pretreatment did not change isoenzyme activities in the first internode of Tir seedlings under DS conditions. Moreover, we did not determine new isoenzymes in the Tir cultivar. In Kıraç-66 seedlings, GST activity decreased after both DS and H2O2 pretreatment due to decreased GST3 activity compared with controls (Fig. 11A and B). 3.9. Transcript levels of cell wall-related genes encoding extensin-like (TaExtLP) and expansin (TaEXPB23) proteins in the first internode To investigate the role of cell wall-related genes on H2O2-induced first internode elongation, the expression of genes coding for extensin- like protein (TaExtLP) and expansin (TaEXPB23) was determined in the first internode of H2O2-treated wheat cultivars under DS conditions (Fig. 12A and B). Although TaExtLP mRNA expression did not change in the first internode of Tir seedlings germinated under DS conditions, it was induced by H2O2 pretreatment under the same conditions. DPI pre- treatment resulted in a stronger increase in its expression. In Kıraç-66 seedlings, both DS and H2O2 pretreatment resulted in downregulation of TaExtLP mRNA expression, but the expression was upregulated by DPI pretreatment under DS conditions. TaEXPB23 was upregulated 2.32-fold in the first internode of Tir following H2O2 pretreatment under shallow-sowing conditions compared to controls. Under DS conditions, TaEXPB23 was down- regulated 5.55-fold, but its expression was upregulated 2.26-fold, by H2O2 pretreatment of the Tir cultivar under the same conditions. The highest expression of TaEXPB23 was obtained for the first internode of H2O2-treated Kıraç-66 seedlings under shallow-sowing conditions, which was 6.14-fold upregulated. Moreover, TaEXPB23 was upregu- lated 2.8-fold in the first internode of Kıraç-66 under DS conditions, but its expression seemed to be more active after seeds were imbibed in H2O2 solution. DPI treatment did not result in any significant change in expression of the TaEXPB23 gene. 4. Discussion Priming of seeds or seedlings with H2O2 activates their antioxidant mechanisms to prevent diverse abiotic stress caused by salinity, drought, heat, or cold. Seed priming by H2O2 has been shown to increase both salt and drought tolerance by enhancing AsA, GSH, and proline levels in Cakile maritima (Ellouzi et al., 2017). Sathiyaraj et al. (2014) reported that H2O2 induced salt tolerance by enhancing the activities of antiox- idant enzymes, such as APX, CAT, and guaiacol peroxidase (POX) in Panax ginseng. Brassica juncea plants treated with H2O2 exhibited higher activities of antioxidant enzymes such as APX, CAT, GR, and GST under drought stress (Hossain and Fujita, 2013). Wahid (2007) reported that low penetration of H2O2 to seeds was able to effectively modulate the expression of genes related to protection against oxidative stress when used in low amounts. Given these prior findings, we evaluated the effects of H2O2-induced changes on DS tolerance in terms of first internode elongation, activities of antioxidant enzymes and glutathione-related enzymes, and expression of cell wall genes in both deep-sowing tolerant and deep-sowing sensitive wheat seedlings under DS conditions. In both arid and semi-arid regions, it is challenging for crop plants to obtain sufficient water for seed germination or seedling establishment due to limited rainfall (Suge et al., 1997; Takahashi et al., 2001). Therefore, in these regions, seeds are sown much deeper in the soil and plants elongate their organs, such as the coleoptile, mesocotyl, and first internode, to elevate their apical meristems to the soil surface (Hoshi- kawa, 1969; Takeda and Takahashi, 1999; Chen et al., 2001; Takahashi et al., 2001). Elongation of these organs is an important adaptive strategy for plants to achieve DS tolerance. However, among these or- gans, only the first internode has been studied in detail (Suge et al., 1997; Nishizawa et al., 2002; Chen et al., 2003; Kato et al., 2011). Fig. 8. Activities of (A) catalase (CAT), (B) peroxidase (POX), and (C) ascorbate peroxidase (APX) in the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or diphenyleneiodonium chloride at 2 sowing depths ((2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)). Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the least significant difference test. Fig. 9. Total glutathione content of the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or diphenyleneiodonium chloride at 2 sowing depths [(2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)]. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the least significant difference test. In earlier studies, H2O2 application has been reported to improve growth of root hairs (Chen et al., 2003; Achard et al., 2008), lateral (Su et al., 2006) and adventitious (Li et al., 2009) root development, cole- optile elongation (Schopfer, 1996), pollen tube growth (Potocký et al., 2007) and cell wall development (Potikha et al., 1999). The strongest stimulatory effect was observed when 0.05 μM H2O2 was applied to seeds. However, the described H2O2 pretreatment did not induce first internode elongation when applied to the growth medium (not the imbibition medium) of wheat seeds. Accordingly, we proposed that H2O2 absorbed by seeds might influence the activities of both H2O2-producing/-metabolizing antioxidant enzymes and glutathione-related enzymes and activate the expression of cell wall-related genes in the first internode, leading to ROS regulation and cell wall loosening. H2O2 acts as an important signaling molecule in plants at low con- centrations, activating both antioxidant and glutathione-related en- zymes and regulating cell division and expansion (Carol and Dolan, 2006; Gappes and Dolan, 2006; Turkan, 2018). Schopfer (1996) was the first to show that exogenous application of H2O2 (10–10000 μM) inhibited the elongation of maize coleoptiles due to the H2O2-dependent formation of phenolic cross-links between cell wall polymers, leading to cell wall-stiffening. Soon after this finding, the same author (Schopfer, 2001) reported that excessive H2O2 was able to acquire charge from OH-mediated cell wall-loosening reactions, which led to both hypocotyl and coleoptile elongation. In line with these prior findings, we showed that a low concentration (0.05 μM) of H2O2 increased the first internode length, whereas a high concentration (40000 μM) decreased it, in the DS tolerant wheat cultivar Tir. On the other hand, Liu et al. (2014) showed that H2O2 accumulation in Pisum sativum could directly or indirectly promote cell wall softening reactions, leading to growth of the pod wall. In Tagetes erecta, H2O2 was found to increase the root length (Liao et al., 2009), while in Arabidopsis thaliana it was found to accelerate lateral root formation (Ma et al., 2014). In contrast, Joo et al. (2001) reported that increased extracellular H2O2 content inhibited cellular growth during root gravitropism in Arabidopsis. Jiang et al. (2012) showed that exogenous H2O2 inhibited root gravitropism while inducing curvature of significant increase in endogenous H2O2 content in the first internode of Kıraç-66, but a decrease in Tir seedlings, was observed despite the increased length of the first internodes, as compared with controls (Fig. 5). Our findings demonstrated that (1) insufficiency of H2O2 sup- presses first internode elongation under DS conditions and (2) elonga- tion of the first internode resulted from H2O2 accumulation in cells, which implies that wheat seeds might absorb H2O2, activate biochemical reactions and molecular mechanisms related to H2O2 metabolism, and induce elongation of the first internode. NADPH oxidases (NOXs), which catalyze the production of super- oxide ions (O •-), play important roles in several growth and develop- ment processes (Marino et al., 2012). Accordingly, the present study showed that Tir seedlings, characterized by the longest first internode under DS conditions, had higher NOX activity than Kıraç-66 seedlings. Moreover, pretreatment of seeds with 0.05 μM H2O2 triggered a statis- tically significant increase in NOX activity in the first internode of both cultivars and their length under DS conditions. A previous study re- ported 20% shorter roots in DPI-treated maize than in wild type maize due to decreased ROS production (Liszkay et al., 2004). Similarly, in the knockout mutant of AtRbohF and AtRbohD/F-double mutant of Arabi- dopsis, both plant size and root length are reported to be reduced (Torres et al., 2002; Kwak et al., 2003). Conversely, Jiang et al. (2012) showed that DPI had no effect on root horizontal curvature. In the present study, DPI treatment caused a significant decrease in endogenous H2O2 content as well as a significant increase in NOX activity in the first internodes of both Tir and Kıraç-66 seedlings under DS conditions (Figs. 5 and 6). Additionally, DPI fully inhibited elongation of the first internode of both cultivars under the same conditions. The average first internode length of these seedlings (DS + DPI) was 21% (Tir) and 13% (Kıraç-66) shorter than that of DS plants, respectively, indicating that cell expansion may be defective in DPI-treated plants (Fig. 1B). These findings suggest that NOX-derived ROS accumulation, caused by pretreatment with H2O2, may play a role in the induction of first internode elongation, thereby enabling DS tolerance. Plasma membrane-localized NADPH oxidase is a critical enzyme involved in O2— and H2O2 generation. While SOD catalyzes the dispro portionation of O — into both H O and O , CAT, POX, and APX are the elongation of first internode was determined by measuring the length of the first internode of wheat seedlings germinated under 10 cm DS con- ditions. When seeds were imbibed in dIH2O, level of H2O2 increased in the first internode of the DS tolerant Tir seedlings, which showed the longest first internode. However, the level of H2O2 in the deep-sowing sensitive Kıraç-66 cultivar decreased under DS conditions, which resulted in decreased length of the first internode. Conversely, when seeds were imbibed in 0.05 μM H2O2 and sown under DS conditions, a main scavengers of H2O2 (Mittler, 2002). Therefore, we speculate that, under DS conditions, a high H2O2 content, as detected in the first internode of DS tolerant Tir seedlings, might result from high NOX and SOD activities and low POX and CAT activities. In Kıraç-66, SOD was unchanged, NOX activity decreased, and POX and CAT activities increased, which may lower the H2O2 content in the first internode and suppress elongation of the first internode. Therefore, in contrast to Kıraç-66, H2O2 accumulation in the first internode of Tir seedlings germinated under DS conditions, which coincided with increased SOD and NOX activities and decreased CAT and POX activities, may imply that H2O2 acts as a signaling molecule related to DS tolerance. On the other hand, under the same conditions, exogenous application of H2O2 decreased CAT and POX and increased SOD and NOX activities in Kıraç-66, suggesting that the remarkable induction of endogenous H2O2 levels in Kıraç-66 resulted from a decreased H2O2 scavenging system, leading to the promotion of first internode elongation under DS condi- tions. It is possible that decreased H2O2 content in H2O2-treated Tir under deep-sowing conditions might be related to the significant increment in POX activity or formation of hydroxyl radical from H2O2, which can react with polysaccharides and destroy the cell wall of the first internode cells as it was reported in maize coleoptile by Schopfer (2001). Fig. 10. Activities of (A) glutathione reductase (GR), (B) dehydroascorbate reductase (DHAR), and (C) glutathione peroxidase (GPX) in the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or diphenyleneiodo- nium chloride at 2 sowing depths [(2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)]. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the least significant difference test. Fig. 11. (A) Total activities and (B) isoenzyme patterns of glutathione S-transferase enzyme (GST) in the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or diphenyleneiodonium chloride at 2 sowing depths [(2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)]. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level, according to the least significant difference test. APX, another H2O2 scavenger, has a higher affinity for H2O2 than CAT or POX. It should be noted that both the deep-sowing tolerant Tir and deep-sowing sensitive Kıraç-66 cultivars maintained their APX ac- tivities in the first internode at similar values under DS conditions compared to the control group. Under the same conditions, pretreatment of wheat seeds with H2O2 did not cause any changes in APX activity in either cultivar under DS conditions, but did cause changes in CAT and POX activities. Therefore, in contrast to both POX and CAT, the effect of APX on DS tolerance cannot be associated with H2O2 degradation. Glutathione (GSH), a low molecular weight antioxidant and ubiq- uitous tripeptide present in plant cells, functions as an intracellular redox buffer, a cofactor for antioxidant enzymes, and a modulator of the activity of thiol-dependent enzymes (Klatt and Lamas, 2000; Filomeni et al., 2002). Furthermore, it is involved in diverse physiological pro- cesses including plant growth, signal transduction, cell cycle regulation, cell death, and senescence (Kranner et al., 2006). Several previous studies have demonstrated that root elongation is dependent on the redox status of growing tissues in roots (Co´rdoba-Pedregosa et al., 2003, Tyburski and Tretyn, 2010). For example, Tyburski and Tretyn (2010) reported that root elongation was related to an optimal GSH level (but not GSSG level) in the apical region of roots. Wang et al. (2017) reported that exogenous NO (nitric oxide) caused a significant increase in the GSH level in the root apex of soybean, thus improving root growth inhibition via Al2+. However, the potential roles of glutathione accumulation and glutathione-related enzymes during first internode elongation were not investigated. Additionally, whether seed pretreatment with H2O2 can regulate glutathione-related enzymes such as GR, GPX, and GST and its isoenzymes, as well as total glutathione content, in the first internode of seedlings under DS conditions is not known. We found that DS treatment induced glutathione accumulation and changed the redox status in the first internode of the deep-sowing tolerant Tir cultivar, but not in the deep-sowing sensitive Kıraç-66 cultivar. The total GSH pool was further increased in the first internode of seedlings arising from both H2O2-treated Tir and Kıraç-66 seeds under DS conditions. The length of their first internodes also increased. Hence, both cultivars accumulated more GSH and had high GSH/GSSG ratios in their first internode in shallow-sowing conditions, as compared to the corresponding values in DS conditions. These results suggest that a high GSH/GSSG ratio in the first internode may be essential for the maintenance of DS tolerance. Furthermore, H2O2 was of great importance in increasing glutathione in the first internode, thus improving induction in first internode growth under DS. Fig. 12. Expression of the genes coding for extensin-like protein (TaExtLP) and expansin (TaEXPB23, RT23) in the first internode of 70 seedlings arising from Tir and Kıraç-66 wheat seeds treated with either H2O2 or diphenyleneiodonium chloride at 2 sowing depths [(2 cm, control, Ctrl) and (10 cm, deep-sowing, DS)]. Expression was normalized using β-actin. Control plants were used as a reference point (set to 1). Error bars indicate the standard deviation of quan- titative reverse transcription polymerase chain reaction runs, each performed in triplicates. Groups that are significantly different from the control group, according to Student’s t-test, are indicated by *, P < 0.05. GR and DHAR, which are prominent components of the Asada- Halliwell-Foyer (AsA-GSH) cycle, are important for maintaining the redox status of both glutathione and ascorbate pools in plant cells (Noctor et al., 2012). Our results demonstrated that the activities of both enzymes significantly increased in the first internode of the Tir cultivar in response to DS; however, in the Kıraç-66 cultivar, GR decreased while DHAR increased. Pretreatment of wheat seeds with 0.05 μM H2O2 caused further increase in GR activity in the first internode of both cultivars under DS conditions. This increment can explain the observed increase in GSH/GSSG ratio (Figs. 9 and 10). These results primarily suggest that the H2O2-induced increase in GR activity contributes to the maintenance of a high GSH/GSSG ratio in the first internode under DS conditions to prevent GSH depletion. Enzymes that use glutathione as a substrate, such as GST and GPX, have important cellular functions. However, their activities in respect to DS have not been elucidated previously. Roxas et al. (1997) reported that overexpression of GST/GPX stimulated growth of tobacco seedlings under both chilling and salt stress and showed that improvement in growth depended on increased GSH pool oxidation, which resulted from the scavenging of excessive H2O2 by GPX. In our study, the first in- ternodes of Tir seedlings under DS conditions showed a 42% increase in GST activity compared to control plants. Moreover, pretreatment of wheat seeds with 0.05 μM H2O2 contributed to maintaining this increase in GST activity under DS conditions. Although GST activity decreased in response to DS in the Kıraç-66 cultivar, it reached the control level with H2O2 pretreatment under DS conditions. The length of the first internode of H2O2-pretreated Tir and Kıraç-66 seedlings, which showed high GST activity, was increased in response to DS. Likewise, due to high GST activity, the first internode of seedlings of both cultivars germinated under DS conditions accumulated more GSSG than control plants. Changes in GSH/GSSG accumulation in the first internode of H2O2-pretreated Tir and Kıraç-66 seedlings may alter redox-sensitive regulatory proteins that control the activities of enzymes critical for first internode processes, such as germination and seedling growth, as reported for tobacco by Roxas et al. (1997). These results indicate that the H2O2-mediated increase in GST activity might promote growth of the first internode under DS conditions by influencing glutathione ho- meostasis. Similar to the findings for GST, the H2O2-mediated increase in GPX activity led to more rapid accumulation of GSSG in the first internode of Kıraç-66 seedlings under DS conditions, which may have caused elongation of the first internode. These results suggest that both GPX and GST act as signaling molecules during elongation of the first internode under DS conditions via regulation of the GSH pool and al- terations of redox-sensitive regulatory proteins that control the enzymes responsible for first internode growth. Two cell wall-loosening processes have been reported: (i) breakage of covalent bonds among cross-linked polymers and (ii) disruption of noncovalent bonds, such as hydrogen or ionic bonds, between cell wall polymers. McQueen-Mason and Cosgrove (1994) showed that expansins cause cell wall loosening by disrupting hydrogen bonds between cellu- lose microfibrils and matrix polymers. Therefore, the expression of expansin genes is correlated with cell elongation. Extensins are the most abundant protein group in the cell walls of higher plants and play roles in various growth and development processes, such as embryonic development (Velasquez et al., 2011) and root hair growth (Pereira et al., 2011; Velasquez et al., 2012). Keskin (2019) demonstrated that the TaExtLP gene, which codes an extensin-like protein in wheat, is upregulated under drought stress. This gene was found to play an important role in plant defense by strengthening the cell wall to prevent tissue damage. In the present study, only the expression of TaEXPB23, which codes for expansin, was induced by H2O2 pretreatment in the first internode of both cultivars under DS conditions, while DPI treatment suppressed its expression under the same conditions. Although DPI treatment upregulated the expression of TaExtLP, H2O2 pretreatment did not result in any decrease in its expression in the first internode of either cultivar under DS conditions. These results indicate that growth of the first internode of seedlings arising from H2O2-treated seeds under DS conditions might be partially influenced by the increased expansin amount, which allows the cell wall loosening necessary for organ elongation. However, detailed experiments and analyses should be conducted to understand the functions and the effect of H2O2 on cell wall loosing processing in plants under deep-sowing conditions. 5. Conclusions There are three main conclusions of this study: (1) H2O2 accumula- tion in the first internode of seedlings arising from H2O2-treated seeds under DS conditions coincided with both increased SOD and NOX ac- tivities and decreased CAT and POX activities, which implies that H2O2 acts as a signaling molecule promoting first internode growth by regu- lating H2O2 generation; (2) H2O2 is of great importance in GSH ho- meostasis, which may activate growth of the first internode to enable DS tolerance; and (3) H2O2 might act as a signaling molecule that upregu- lates the expression of TaEXPB23, the gene coding for expansin, during the first internode elongation. Finally, the ameliorated characteristics of first internode growth, reflected as maintenance of redox status, enhanced capacity of antioxidant enzymes, and expression of cell wall genes, are important indicators of the beneficial effects of seed pre- treatment with H2O2 under DS conditions. Author contributions A.H.S.C., Y.A., I.T. designed the experiment. A.H.S.C., T.Y., Y.A., and A.G. performed the experimental assays and analyzed the data. A.H.S.C. interpreted the data and wrote the manuscript. T.Y., Y.A., A.G., and I.T. carefully proofread the manuscript. Funding This work was financially supported by The Scientific and Techno- logical Research Council of Turkey (TUBITAK), [Project No 114Z034] and the Ege University Research Foundation, [Project No 13-FEN-049]. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors wish to thank Prof. Dr. Muzaffer Tosun from Ege Uni- versity, Faculty of Agriculture, for providing the Triticum aestivum Tir cv. seeds. References Achard, P., Gong, F., Cheminant, S., Alioua, M., Hedden, P., Genschik, P., 2008. 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