Two local rice cultivars with different levels of alkaline tolerance were used in this study: ‘Dongdao-4’ (D-4, moderately tolerant to SA) and ‘Jiudao-51’ (J-51, sensitive to SA) (Feng et al., 2016). D-4 is an elite cultivar in the local SA land area, which was bred by crossing ‘Akitakomachi’ with ‘Nongda-10’ in Da’an Sodic Land Experiment Station, Jilin, China (Yang et al., 2011). The cultivar J-51 was bred by crossing ‘Tong-35’ with ‘Nongda-3’ in Rice research institute, Jilin city Academy of Agriculture Science, Jilin, China (China Rice Data Center).

Seeds were surface-sterilized with 75% (v/v) alcohol for 5 min, and rinsed with deionized water five times. After immersing in water for 2 days, the seeds were sprinkled onto wet filter paper in a Petri dish, and germinated for 1 day at 28°C in a dark incubator. Eighteen uniformly germinated seeds were transplanted onto a multi-well plate floating on a 300-ml cup containing deionized water for 7 days, and then grown for 7 days in half-strength Kimura B nutrient solution (Miyake and Takahashi, 1983) in a controlled growth chamber under the following conditions: 25°C day/20°C night, 12-h photoperiod, and 350 μmol photons m^-2 s^-1 light intensity.

We used Na₂CO₃ at 15 mM (pH = 10.87, EC = 2.7 mS/cm) to simulate alkaline stress. In our previous study, a series of Na2CO3 concentrations ranging from 0 to 50 mM was examined in correlation with cell injury and plant growth inhibition (Lv et al., 2013). Based on results of the study, we chose the lowest Na₂CO₃ concentration (15 mM) where a significant root damage and plant growth inhibition were observed during the experimental period (7 days).

Two-week-old rice seedlings were treated with or without 15 mM Na₂CO₃ solution for 3 days prior to all analyses.

The procyanidins are a group of polyphenolic compounds that exhibit high ROS-scavenging activity, and naturally occur in fruits, vegetables, nuts, seeds, flowers, and bark (Fine, 2000; Rue et al., 2017). Grape seed is a rich source of procyanidins, and grape seed extract (GSE) is widely used as an antioxidant dietary supplement. In this study, procyanidins (98% pure; Ci Yuan Biotechnology Co. Ltd., Shanxi, China) from GSE was used to assess the effect of ROS elimination on alkalinity-induced root damage and seedling growth. Two-week-old rice seedlings were pretreated for 24 h with 0.98% procyanidins prepared in water, followed by treatment with or without 15 mM Na₂CO₃ solution for 3 days.

After 3 days of Na₂CO₃ treatment, the survival rate of the rice seedlings was determined. Ten rice seedlings were randomly selected from the 25 seedlings in each treatment group and scanned using an Epson Expression 10000XL (Epson America Inc., Long Beach, CA, United States). The resulting images were digitized using the WinRHIZO program, according to the manufacturer’s instructions (Regent Instruments Canada Inc., Ville de Québec, QC, Canada), and the average shoot length (SL, the length of the longest leaf), total root length (TRL), total root surface area (RSA), root diameter (RD), total root volume (RV), and root number (RN) were determined. After scanning, the seedlings were cut and divided into shoots and roots, and their fresh weights measured. These samples were then dried in a forced-air-driven oven at 105°C for 2 h, followed by 70°C, until a stable mass was reached, prior to the determination of dry mass. The roots were rinsed four times with deionized water and then dried. Individual seedlings were classified as dead if all the leaves were dry and brown.

Membrane injury (MI) was measured by electrolyte leakage (Tantau and Dörffling, 1991). Ten seedlings were randomly selected from each treatment group, washed with deionized water to remove surface-adhered electrolytes, and cut and divided into shoots and roots. Samples (2 g fresh weight) of the shoots and roots were submerged in 15 ml of deionized water in 50-ml conical tubes and kept at 20°C for 1 h. The electrical conduction of the effusion was then measured (R1). The tissue samples were killed by heating tubes in a boiling bath for 40 min, cooled to 20°C, and the electrical conduction of the effusion was measured again (R2). The MI was evaluated using the formula MI (%) = R₁/R₂ × 100%.

Malondialdehyde (MDA) is a decomposition product of polyunsaturated fatty acid hydroperoxides, and is often used as an indicator of lipid peroxidation due to oxidative stress. The MDA content was determined by the thiobarbituric acid reaction as described by Heath and Packer (1968). A fresh root sample (0.1 g) was homogenized in 1 ml of 50 mM phosphate buffer (pH 7.8) using a bench-top ball-mill (Scientz-48, Ningbo Scientz Biotechnology Co. Ltd., Ningbo, China) at 50 Hz for 30 s, and centrifuged at 12,000 rpm for 15 min. Subsequently, 400 μl of supernatant was mixed with 1 ml of 0.5% thiobarbituric acid, and the mixture was placed in a boiling water bath for 20 min. The mixture was then cooled and centrifuged, and the absorbance of the resulting supernatant was measured at 532, 600, and 450 nm. The MDA content was calculated using the following formula: 6.45 × (A₅₃₂ - A₆₀₀) - 0.56 × A₄₅₀.

Root α-naphthylamine-oxidizing activity was used as an indicator of root vigor according to the method described by Ramasamy et al. (1997). Rice roots (0.1 g) were harvested in a 10-ml tube containing 5 ml of 0.1 mol/l phosphate buffer (pH 7.0) and 25 μg/ml of α-naphthylamine and incubated at room temperature for 10 min. A 200-μl aliquot of the solution was then taken as the reaction control. The tube was further incubated at 25°C for 1 h, and a 200-μl aliquot of the solution was taken, mixed with 1 ml of distilled water, 100 μl of 1% sulfanilic acid anhydrous, and 100 μl of 1 mg/ml sodium nitrite, and incubated for 5 min. After diluting the mixture with 1.1 ml of distilled water, the resulting color was measured using a spectrophotometer at 510 nm. Root vigor is expressed as micrograms α-NA per gram fresh weight (FW) per hour (μg α-NA g^-1 FW h^-1).

Root cell viability was determined by Evan’s Blue staining (Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006). Root tips (2 cm) were infiltrated with a 0.25% (w/v) aqueous solution of Evan’s Blue for 30 min, before being rinsed with deionized water until no further blue dye was eluted from the roots. The stained root tips were observed under a stereoscopic microscope (SMZ1270i, Nikon, Tokyo, Japan). To quantify the Evan’s Blue staining, five stained root tips were solubilized by incubating in 1% (w/v) SDS in 50% (v/v) methanol at 50°C for 30 min, and the absorbance was measured at 600 nm.

The H₂O₂ and O2•- contents were measured using a H₂O₂ and O2•- content determination kit, according to the manufacturer’s instructions (Comin Biotechnology, Suzhou, China).

For the determination of antioxidant enzyme activity, fresh roots (0.1 g) were loaded into a 2-ml tube and frozen in liquid nitrogen, then homogenized in 1 ml of 50 mM phosphate buffer (pH 7.8) using the bench-top ball-mill at 50 Hz for 30 s. The homogenate was centrifuged at 12,000 rpm for 15 min at 4°C, and the resulting supernatant was used for SOD, CAT, and POD assays.

Superoxide dismutase (EC 1.15.1.1) activity was determined using the nitro blue tetrazolium (NBT) method as described by Giannopolitis and Ries (1977). One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction, as monitored at 560 nm. CAT (EC 1.11.1.6) activity was measured according to the method described by Aebi (1984), and was assayed by the decline in absorbance per minute at 240 nm as a consequence of H₂O₂ consumption. POD (EC 1.11.1.7) activity was determined by assessing the rate of guaiacol oxidation in the presence of H₂O₂. One unit of POD was defined as the increase in absorbance per minute at 470 nm (Klapheck et al., 1990).

To assay APX activity, samples were extracted with 1 ml of 50 mM phosphate buffer (pH 7.0) containing 1 mM AsA and 1 mM ethylenediaminetetraacetic acid, and homogenized using the bench-top ball-mill at 50 Hz for 30 s. The homogenate was centrifuged at 12,000 rpm for 15 min at 4°C. Subsequently, the supernatant was mixed with phosphate buffer (pH 7.0), 15 mM AsA, and 0.3 mM H₂O₂. The reaction mixture was analyzed using a spectrophotometer at 290 nm. One unit of APX was defined as the variable quantity of absorbance per minute at 290 nm.

Leaf samples were extracted using a 5-ml mixture of ethanol and acetone (V:V = 1:1). The absorbance of the supernatant was determined at 645 and 663 nm. Total chlorophyll content was calculated using the following formula: 8.02OD_663nm + 20.21 OD_645nmV/1000W.

Roots (0.2 g) were sampled in liquid nitrogen and ground using the bench-top ball-mill at 50 Hz for 30 s. Total RNA was extracted with TRIzol reagent (TaKaRa Bio, Tokyo, Japan), and first-strand cDNA was synthesized using M-MLV reverse transcriptase (Thermo Fisher Scientific, Carlsbad, CA, United States), according to the manufacturer’s protocols. A qRT-PCR was performed to determine the transcriptional expression of five genes related to cell death. Gene-specific primers were designed using Primer 5.0 software (Table 1). The housekeeping gene β-actin (GenBank ID: X15865.1) was used as an internal standard. The PCR was conducted in a 20-μl reaction mixture containing 1.6 μl of cDNA template (50 ng), 0.4 μl of 10 mM specific forward primer, 0.4 μl of 10 mM specific reverse primer, 10 μl of 2× SYBR^® Premix Ex Taq (TaKaRa Bio), and 7.6 μl of double-distilled H₂O in a PCRmax machine (Eco TM 48, Illumina, Saffron Walden, United Kingdom). The procedure was performed as follows: 1 cycle for 30 s at 95°C, 40 cycles for 5 s at 95°C and 20 s at 60°C, and 1 cycle for 60 s at 95°C, 30 s at 55°C, and 30 s at 95°C for a melting curve analysis. The relative expression level was computed using the 2^-ΔΔC_T method (Livak and Schmittgen, 2001).

All of the experiments were conducted in a controlled growth chamber with three biological replicates, each consisting of five to ten plants. Statistical analyses were performed using the statistical software SPSS 21.0 (IBM Corp., Armonk, NY, United States). A one-way analysis of variance (ANOVA) was used to compare the treatment means (P = 0.05). SigmaPlot 10.0 (Systat Software Inc., San Jose, CA, United States) was used for graphical presentation of the data.

Alkaline treatment for 3 days caused the wilting and death of seedlings of both cultivars (Figure 1A). The seedling survival rates under alkaline stress were reduced to 52.3 and 19.3% of the control treatment in D-4 and J-51, respectively (Figure 1B). Shoot FW, shoot dry weight (DW), shoot relative water content (RWC), root FW, and root DW were significantly reduced (Figures 2A–E). In addition, TRL, RSA, TRV, RN, and SL were significantly reduced in both cultivars as compared to the control treatment (Figures 3A,B,D–F). A smaller effect was observed on root diameter (Figure 3C). Overall, alkaline stress appeared to have more deleterious effects on the seedling growth of J-51 than that of D-4.

Root cell MI was assessed by the determination of MI (electrolyte leakage) and MDA content (lipid peroxidation). Alkaline treatment caused severe cell membrane damage in roots as shown by an increase in both MI (Figure 4A) and MDA content (Figure 4B), although the increase in MDA content in D-4 was not statistically significant (Figure 4B). MI and MDA induction under alkaline stress were higher in J-51 than in D-4.

Alkaline treatment markedly decreased root vigor in both cultivars as assessed by α-naphthylamine-oxidizing activity (Figure 5A), and the root vigor in J-51 was inhibited to a greater degree than that in D-4. In addition, strong Evan’s Blue staining of root tips was observed following alkaline treatment (Figure 5B). Measurement of Evan’s Blue levels extracted from the root tips showed a three- and two-fold increase in response to alkaline treatment in J-51 and D-4, respectively (Figure 5C). These results indicate severe root cell damage following alkaline stress.

To gain further insights into cellular damage in roots under alkaline stress, the transcription levels of five genes associated with cell death were analyzed: OsKOD1, an inducer of programmed cell death (PCD) (Blanvillain et al., 2011); OsHsr203j, which is often used as a cell death marker (Pontier et al., 1998; Wakasa et al., 2011); OsCP1, an executor of PCD (Lee et al., 2004; Li et al., 2011); OsNAC4, a NAC domain-containing transcription factor involved in cell death (Kaneda et al., 2009); and OsBI1, a cell death suppressor, which plays important roles in the modulation of PCD in response to abiotic and biotic stress (Weis et al., 2013). As shown in Figure 6, the expression of the cell death-associated genes OsKOD1, OsHsr203j, OsCP1, and OsNAC4 was upregulated 12-, 4-, 16-, and 2-fold, respectively, in response to alkaline stress in J-51 (Figures 6A–D). Similarly, in D-4, these genes were all upregulated in response to alkaline stress (Figures 6A–D), although no statistical significance was observed for OsKOD1 and OsNAC4 (Figures 6A,B). Conversely, the cell death-suppressor gene OsBI1 was significantly downregulated in both cultivars under alkaline stress.

To investigate the possible mechanism of root damage under alkaline stress as shown in Figures 4–6, we determined the O2•- and H₂O₂ contents. The results showed a remarkable accumulation of O2•- (Figure 7A) and H₂O₂ (Figure 7B) in response to alkaline treatment in both cultivars. The levels of O2•- and H₂O₂ accumulation under alkaline stress were much higher in J-51 than in D-4.

In response to stress-triggered ROS accumulation, the plants activated antioxidant defense mechanisms to eliminate excess ROS from their cells. As shown in Figure 8, the activity of the antioxidant enzymes SOD, POD, CAT, and APX were significantly increased in roots in response to alkaline stress; however, the increase in APX activity in D-4 was not statistically significant (Figure 8D). The activity of all of the antioxidant enzymes tended to be higher in D-4 than in J-51 plants.

To investigate the association between root damage, the alkalinity-induced inhibition of plant growth (Figures 1–6), and ROS accumulation (Figure 7), we applied exogenous GSE that contained 98% natural procyanidins, which is a potent antioxidant. Exogenous GSE significantly reduced seedling wilt (Figure 9A), chlorophyll loss (Figure 9B), cell MI (Figure 9C), and cell death (Figure 9D) concomitantly with reduced ROS accumulation (Figures 9E,F) under alkaline stress.