Improved waterlogging tolerance of barley (Hordeum vulgare) by pretreatment with ethephon

Root growth into hypoxic or anoxic waterlogged soil relies on internal aeration in plants. The plant hormone ethylene helps adapt to waterlogging by inducing the formation of aerenchyma, which provides a low-resistance pathway for the transport of oxygen from the shoot to the root apex. Waterlogging-susceptible crops including barley start to form aerenchyma after suffering waterlogging stress. But waterlogging can be fatal if aerenchyma formation is not fast enough. Here, we investigated whether pre-treating barley with ethephon, an ethylene-releasing agrochemical, could improve its tolerance to mimicked waterlogging conditions (using stagnant deoxygenated agar nutrient solution). In barley growing in aerated nutrient solution, ethephon treatment enhanced aerenchyma formation at the root tips and induced the development of shorter and shallower roots. Pre-treating barley leaves also delayed waterlogging-caused whiting and increased the percentages of viable root-tips under waterlogging conditions. However, the pretreatment did not noticeably increase fresh weight or shoot length. Further studies are needed to optimize ethephon treatment conditions to improve barley production under waterlogged conditions. ARTICLE HISTORY Received 17 July 2018 Revised 25 December 2018 Accepted 7 February 2019


Introduction
Plants can suffer from hypoxia or even anoxia when soils become waterlogged. Well-drained soil is porous and normally filled with gas; excess water fills the pores, preventing the entry of atmospheric oxygen, as the diffusivity of oxygen in water is extremely low (Jackson, Fenning, Drew & Saker, 1985). Other problems associated with waterlogging are the accumulation of phytotoxic compounds in the soil (Ernst, 1990;Kreuzwieser, Papadopoulou & Rennenberg, 2004;Lamers, Tomassen & Roelofs, 1998;Ponnamperuma, 1984) and a decline in the availability of some nutrients (Laanbroek, 1990).
Ethylene is not easy to use in experimental studies because it is a gas. Instead, researchers often use 1-aminocyclopropanecarboxylic acid (ACC), which is a metabolic intermediate of ethylene and is degraded into ethylene by the activity of ACC oxidase in plants (Roblin & Pérault, 1985;Zhang & Wen, 2010). Recently, pre-treating wheat with ACC before exposing it to waterlogging-like conditions was shown to improve growth, stimulate aerenchyma formation and upregulate the expression of ethanol fermentation enzymes in first seminal roots (Yamauchi et al., 2014). Another ethylene-producing chemical is ethephon. It is widely used in horticultural crops to improve lodging resistance, ripen fruit and stimulates flowering (Gent & McAvoy, 2000). It releases ethylene over a long period as it is chemically degraded (Maynard & Swan, 1963;Peng, Chan, Shih & Yang, 2001;Roblin & Pérault, 1985;Zhang & Wen, 2010). Here, we investigated whether ethephon pretreatment could also improve waterlogging tolerance. Whereas other studies of ACC pretreatment mainly focused on adaptive responses in single roots, we mainly focused on adaptive responses in whole root system under waterlogged conditions.

Growth conditions
Seeds of barley (Hordeum vulgare cv. Morex) were kindly provided by the Genetic Resources Center, National Agriculture and Food Research Organization, Japan (https://www.gene.affrc.go.jp/index_en.php). Barley plants were grown in the same nutrient solution used in earlier studies (Colmer, 2003;Shiono et al., 2011). Plants were grown in a controlled-environment chamber (20-h light at 23°C/4-h dark at 23°C, relative humidity over 50%, photosynthetic photon flux density at 249 μmol m −2 s −1 ). Seeds were sterilized for 30 min in 0.6% (w/v) sodium hypochlorite, washed thoroughly with deionized water, and for imbibition, were placed in Petri dishes containing about 5 mm of deionized water for 1 day in darkness at 23°C. For germination, we used a tank containing aerated, quarter-strength nutrient solution. A stainless mesh was placed in the tank so that its upper surface was at the water level. The mesh pores sized smaller than seeds but large enough for root growth. One day after imbibition, seeds were placed on the mesh and exposed to light. For the ethephon treatment, we prepared 5-liter pots (120 mm × 180 mm × 250 mm high) containing aerated full-strength nutrient solution containing 0, 0.1, 1 or 37.5 µM ethephon (Nissan Ethrel®10, Nissan Chemical Industries, Tokyo, Japan). Following Steffens, Kovalev, Gorb and Sauter (2012), we used ethephon treatments of 0, 37.5, 70 and 150 µM. In each pot, a rectangular 2-cm-thick piece of foam was placed on the solution and aluminium foil was placed on the top of the foam to keep the solution dark. In each pot, vertical cuts were made on the six sides of the foam to accommodate stems. Then 6 plants were transferred to each pot, sliding the stems into the cuts. In this way, the root was kept in the dark. Seven days later (13 days after imbibition), plants were transferred to pots containing aerated nutrient solution or stagnant deoxygenated nutrient solution for 7 days. The stagnant solution was prepared by adding 0.1% (w/v) agar to the nutrient solution and boiling the solution to dissolve the agar. The low concentration of agar produced a viscous liquid rather than a gel. By preventing convective movements, the solution mimics the changes in gas composition found in waterlogged soils (e.g. decreased oxygen and increased ethylene) (Wiengweera, Greenway & Thomson, 1997). The solution was poured into the pots and deoxygenated by bubbling N 2 gas through two air stones at a flow rate of about 2.2 L min −1 for 15 min per pot. The dissolved oxygen level was confirmed to be less than 1.0 mg L −1 by dissolved-oxygen meter (SG6-ELK, Mettler Toledo, Greifensee, Switzerland).

Growth parameters
Plants were harvested before waterlogging treatment (i.e. after pretreatment) and after 7 days of waterlogging treatment under stagnant conditions. Shoot fresh weight (FW), shoot length, leaf age, longest root length and total root number were recorded for each plant. Chlorophyll content of leaves was measured using a Soil Plant Analysis Development (SPAD) meter (SPAD-502Plus, Konica Minolta, Tokyo, Japan). The chlorophyll content was read at the middle of a leaf. During the 7 days of waterlogging treatment only the 1st leaf (the oldest leaf) was read. After 7 days of waterlogging treatment (i.e. when the seedlings were 20 dayold), all leaves (i.e. 1st, 2nd and 3rd leaves) were read.

Root porosity
Root porosity (P) is a measure of the gas volume in the roots: where V g is the gas volume in the roots (including aerenchyma and intercellular space) and V r is the volume of the root tissue. It is measured by determining root buoyancy before and after vacuum infiltration of water into the gas spaces in the roots (Katayama, 1961;Raskin, 1983;Thomson, Armstrong, Waters & Greenway, 1990). Roots were cut into 50 mm segments. All of the segments from one plant were combined, gently blotted to remove excess water and weighed (w1). Three paper clips were used to hold the segments together and act as a sinker. Using an underhook balance (PA213CJP, Ohaus corporation, New Jersey, USA), which weighs objects suspended below the balance, we measured the weight of just the paper clips hanging from a metal hook below the balance in a 2-L beaker of water (w2) and the weight of the roots held with the same paper clips in the water (w3). A smaller beaker containing the roots and paper clips in the water was placed in a vacuum desiccator and subjected to three 5-min periods of light vacuum to release the gas in the roots and infiltrate the roots with water. Finally, the paper clips and infiltrated roots were weighed in water (w4). Following Thomson et al. (1990), the above equation can then be expressed as

Aerenchyma formation
Adventitious roots (8-15 cm length) were cut at the root-shoot junction. Formation of aerenchyma was measured in cross-sections at 15-20 mm from the apex. Parts of the roots were collected and stored in deionized water at 4°C for no more than 4 days before observation. Fresh roots were sliced by hand under a stereomicroscope (Leica S9D, Leica Biosystems, Wetzlar, Germany). The cross-sections were viewed with an Axio Imager A2 microscope and photographed with an AxioCam MRc camera (both Carl Zeiss, Oberkochen, Germany). The proportion of each cross-section occupied by aerenchyma was determined using Image J software (Ver. 1.50i; NIH, Bethesda, USA).
TTC reduction assay 2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) is a colourless compound that turns red when reduced by dehydrogenases in living cells. TTC (WAKO, Osaka, Japan) was dissolved in 0.1 M sodium phosphate buffer (pH 7.0) to a final concentration of 0.6% (w/v). To determine whether a barley root was alive, the root was cut at the root-shoot junction, incubated in TTC solution at 40°C for 30 min, rinsed in deionized water and scanned with a scanner (Colour mode, 400 dpi; EPSON Perfection V800 Photo, SEIKO EPSON, Suwa, Japan). Root tips that turned red were considered to be alive. The percentage of viable root-tips (V) was taken as the per cent of roots that were alive: where a is the number of root-tips turned red by TTC staining and n is the root numbers.

Statistical analysis
In comparisons of more than three groups, the means were compared with one-way ANOVA and Tukey HSD tests for multiple comparisons at the 5% probability level. The data were analysed with IBM SPSS Statistics version 25 (IBM, New York, USA). Differences in percentage of viable root-tips were compared with Fisher's exact test at the 5% probability level. The data were analysed with R version 3.5.0 (R Core Team, 2018) (R packages: pwr).

Effects of ethephon on morphology and growth
After ethephon pretreatment, aerenchyma formation (% cross-section) was significantly enhanced at 2 cm from the root apex (P < 0.05, Figure 1(a)). Ethephon pretreatments at concentrations of 0.1, 1 and 37.5 μM resulted in aerenchyma occupying 6.7%, 8.2% and 11.3% of the cross-sections, respectively. The percentages of aerenchyma were 2.2% without ethephon pretreatment. Thus, pretreatment with ethephon at concentrations of 0.1, 1 and 37.5 μM increased aerenchyma formation 3.0, 3.7 and 5.1 times, respectively. Ethephon pretreatment at concentrations of 70 and 150 µM remarkably reduced root porosities (data not shown), so that we used 37.5 µM as the maximum concentration. Root porosities were not significantly enhanced by ethephon pretreatment, being lower than 3% even with 37.5 μM ethephon (P > 0.05, Figure 1(b)). We observed that plants with 1 or 37.5 μM ethephon pretreatment had shallower root systems than did untreated plants (Figure 2). The lengths of the longest roots with 1 or 37.5 μM ethephon were 10 and 13 cm, respectively (Table 1), significantly shorter than the longest roots (21-22 cm) in plants treated with 0 and 0.1 μM ethephon (P < 0.05). However, the ethephon treatments did not significantly affect total root numbers (P > 0.05, Table 1). Thus, these shallow root systems were caused by a reduction of root growth. Treatments of 1 and 37.5 μM ethephon also reduced shoot FW (P < 0.05, Table 1).

Effect of ethephon on waterlogging tolerance
Without ethephon pretreatment, the first (oldest) leaf was dark green. Its chlorophyll content (SPAD value) remained high during the first 3 days in the stagnant agar solution, declined after 4 days and fell to zero at 6 days as the leaf turned yellowish-white (Figure 3). Pretreatment with ethephon, especially at the higher concentrations, slowed the decline in the SPAD value of the first leaf. Pretreatment with 37.5 μM ethephon significantly increased the SPAD value after 5 and 6 days of stagnant treatment (P < 0.05). Thus, ethephon delayed the whiting of the first leaf. At 7 days of stagnant treatment, ethephon reduced the number of yellowish-white leaves (Figure 4(a)). Ethephon also significantly increased the SPAD values in second and third leaves (P < 0.05, Figure 4(b)). Ethephon at 37.5 μM remarkably increased the SPAD values in all leaves.
Regarding growth under stagnant conditions, ethephon pretreatment did not noticeably increase the FW of shoots, shoot length, leaf age or total root Table 1. Growth of barley seedlings after 7 days ethephon pretreatment (13 day-old-seedlings).
number (P > 0.05, Table 2). Without ethephon pretreatment, shoot FW in stagnant conditions was reduced to 61% of the FW under aerated conditions. The reductions of shoot FW with ethephon pretreatment ranged from 56% to 64%, close to the value of untreated plants grown under stagnant conditions. However, the per cent increase in the growth of the longest root lengths during the treatment period was significantly greater in the 1 μM and 37.5 μM ethephon treatments than in the control (P < 0.05, Table 3). This implies that the longest roots kept growing under stagnant conditions.

Root activities under stagnant conditions
After 2 days of stagnant conditions, most of roots treated with 37.5 μM ethephon were still alive, although many inactive roots were observed in the untreated plants ( Figure 5). After 7 days of stagnant treatment, most of the root apexes of the plants treated with 37.5 μM ethephon were still alive (i.e. they turned red with TTC staining), while many of the roots of the untreated plants were inactive (Figure 6(a)). For roots with lengths of 11-15 cm, the percentage of viable root-tips increased dramatically with increasing ethephon concentration (P < 0.05, Figure 6(b)). Few inactive roots were observed (a) Overview of leaf colour. Scale bar = 10 cm. (b) SPAD values of 1st, 2nd or 3rd leaves. Means ± SE. n = 3. Different letters denote significant differences among different concentrations of ethephon pre-treatment (P < 0.05, one-way ANOVA and then Tukey HSD test for multiple comparisons). Plants were grown in aerated nutrient solution for 6 days, and then transferred to aerated nutrient solution for 7 days in the absence or presence of ethephon. Thirteenday-old seedlings were transferred to pots containing aerated nutrient solution or stagnant deoxygenated nutrient solution for 7 days.
in the plants treated with 37.5 μM ethephon. Ethephon pretreatment maintained the viability of roots over 11 cm length under stagnant conditions.

Discussion
Ethylene is a key inducer of adaptive responses to waterlogging including aerenchyma formation, development of new adventitious roots and activation of fermentation pathways (Bailey-Serres & Voesenek, 2008;Shiono et al., 2008). Ethephon pretreatment enhanced aerenchyma development at the root tip before waterlogging, but not at the basal part of root ( Figure 1). Additionally, plants pretreated with ethephon developed a shallow root system and their longest roots were shorter than those of untreated plants (Figure 2, Table 1). Ethylene inhibits root extension (Visser, Nabben, Blom & Voesenek, 1997;Visser & Pierik, 2007). A shallow root system allows plants to grow and survive in waterlogged soil because the infiltration of atmospheric air keeps the oxygen concentration at the soil surface relatively high (Jackson & Drew, 1984;Shiono et al., 2008). For instance, some wetland plants including rice have shallow root systems (Jackson & Drew, 1984;Kawata, Yamazaki, Ishihara, Shibayama & Lai, 1963;Shiono et al., 2008). Wheat cultivars with shallow root system have a higher waterlogging tolerance rather than cultivars with deeper root system (Haque et al., 2012;Oyanagi et al., 2004). Dissolved oxygen levels at depths of 3.28 ± 0.51 a 2.09 ± 0.10 a 64 ± 3 a 37.5 3.20 ± 0.67 a 1.78 ± 0.08 a 56 ± 3 a Shoot length (cm) 0 3 6 ± 1 a 32 ± 1 b 90 ± 3 ab 0.1 34 ± 2 a 32 ± 1 b 92 ± 4 b 1 3 3 ± 2 a 31 ± 1 b 93 ± 2 b 37.5 30 ± 1 a 24 ± 1 a 79 ± 2 a Leaf age 0 4.8 ± 0.2 a 3.8 ± 0.2 a 79 ± 4 a 0.1 5.0 ± 0.2 a 3.9 ± 0.0 a 78 ± 1 a 1 5.4 ± 0.3 a 3.9 ± 0.0 a 73 ± 0 a 37.5 5.5 ± 0.2 a 3.9 ± 0.1 a 71 ± 1 a Longest root length (cm) 0 5 0 ± 2 b 18 ± 1 a 37 ± 2 a 0.1 47 ± 3 ab 18 ± 2 a 38 ± 3 a 1 4 1 ± 2 ab 15 ± 1 a 36 ± 2 a 37.5 36 ± 3 a 13 ± 1 a 36 ± 2 a Total root number 0 1 9 ± 2 a 20 ± 1 b 104 ± 3 b 0.1 19 ± 2 a 20 ± 2 b 106 ± 11 b 1 1 7 ± 2 a 19 ± 1 b 111 ± 7 b 37.5 19 ± 2 a 13 ± 1 a 69 ± 3 a Mean ± SE. n = 3-6. Different superscript letters denote significant differences among concentrations of ethephon treatments (P < 0.05, one-way ANOVA and then Tukey HSD test for multiple comparisons). § Per cent controls were calculated by each growth in aerated or stagnant solution with each concentration of pretreatment. Plants were grown in aerated nutrient solution for 6 days, and then transferred to aerated nutrient solution for 7 days in the absence or presence of ethephon. Thirteenday-old seedlings were transferred to pots containing stagnant deoxygenated nutrient solution for 7 days. Mean ± SE. n = 3. Different superscript letters denote significant differences among concentrations of ethephon treatments (P < 0.05, one-way ANOVA and then Tukey HSD test for multiple comparisons). Plants were grown in aerated nutrient solution for 6 days, and then transferred to aerated nutrient solution for 7 days in the absence or presence of ethephon. Thirteen-day-old seedlings were transferred to pots containing stagnant deoxygenated nutrient solution for 7 days. § Per cent increases were calculated from each value at the commencement day of stagnant treatment (13-day-old seedlings) and the end day of the treatment (20-day-old seedlings). Viable root tips are indicated by red TTC staining. The number of viable root-tips is expressed as a percentage of the total number of roots. Means ± SE. n = 3. Different letters denote significant differences among four different concentrations of ethephon pre-treatment (P < 0.05, one-way ANOVA and then Tukey HSD test for multiple comparison). Plants were grown in aerated nutrient solution for 6 days, and then transferred to aerated nutrient solution for 7 days in the absence or presence of ethephon. Thirteen-day-old seedlings were transferred to pots containing stagnant deoxygenated nutrient solution for 2 days.
1, 5 and 15 cm in the stagnant deoxygenated agar solutions after 7 days were 4.9 ± 0.1, 1.5 ± 0.2 and 0.3 ± 0.0 mg/ L (mean ± SE), respectively. The oxygen concentrations were higher at the surface of stagnant solutions than at the bottom (Supplemental Table 1). Under waterlogged conditions, longer roots (> 10 cm) were inactive (TTC-negative), but relatively short roots (≤ 10 cm) were still active without ethephon pretreatment (Figure 6(b)). The shallow root system in treated plants may contribute to waterlogging tolerance. Additionally, ethephon pretreatment enhanced aerenchyma formation (Figure 1(a)). A mathematical simulation (Armstrong, 1979) predicted that non-aerenchymatous roots would be limited to a depth because of the difficulty of respiration at the root tips, while aerenchymatous roots could go deeper. Enhancement of aerenchyma formation and shallow root system by ethephon pretreatment might maintain the activities of root tips under waterlogged conditions (Figures 4,5), which could explain the delay of leaf chlorosis during waterlogged treatments (Figures 2,3). Pre-treating wheat with ACC, an ethylene precursor, was also found to improve its waterlogging tolerance (Yamauchi et al., 2014). However, there are a few dissimilarities between the present results and the previous report. In this study, the only effect of ethephon on barley growth was that it increased the length of the longest roots (Table 3). The growth parameters on shoot growth (i.e. Shoot FW and shoot length) were not improved by ethephon pretreatment (Tables 2,3). On the other hand, in wheat, 2-days of pretreatment using 20 µM ACC, an ethylene precursor, enhanced not only shoot length and shoot dry weight but also the lengths of the longest roots (Yamauchi et al., 2014). Different responses to ACC and ethephon were sometimes observed in other plants and tissues (Lavee & Martin, 1981). The effects of ACC were limited to the treated tissues. Additionally, exogenously applied ACC failed to have prolonged effects (Zhang & Wen, 2010). Ethephon can release ethylene over longer periods. However, it may also cause non-specific responses (Zhang & Wen, 2010). Another difference is that when ethephon is degraded in aqueous solution, it produces toxic acids (e.g. phosphoric acid and hydrochloride) (Biddle et al., 1976;Goudey, Saini & Spencer, 1987).
Although ethephon pretreatment did not significantly increase growth in this study, it prolonged the survival of barley for a few days under waterlogging conditions. Further studies are needed to optimize the period, growth stage and concentrations of ethephon treatment.