Seeds of peanut cultivars 99-1507 (Al-tolerant) and ZH2 (Al-sensitive) were prepared as described by Zhan et al. (2008) with slight modifications. After incubating in moistened perlite for 3–4 days in the dark at 26 ± 1°C, seedlings with roots 2–3 cm long were transferred to Hoagland nutrient solution, which was renewed every 2 days with fresh nutrient solution. After the emergence of the fourth leaf, the seedlings were pretreated for 24 h in 0.1 mM CaCl₂ (pH 4.2) solution. In the time-course treatment, the seedlings were exposed to 100 μM AlCl₃ (containing 0.1 mM CaCl₂, pH 4.2) for 0, 4, 8, 12, 24h, respectively. For elucidating the mechanism of NO on alleviating Al toxicity, seedlings were exposed to the following treatments for 12 h as follows: 100 μM AlCl₃ (containing 0.1 mM CaCl₂, pH 4.2); 100 μM AlCl₃ and 200 μM SNP (containing 0.1 mM CaCl₂, pH 4.2); 100 μM AlCl₃ and 50 μM cPTIO (containing 0.1 mM CaCl₂, pH 4.2); 200 μM SNP (containing 0.1 mM CaCl₂, pH 4.2); 50 μM cPTIO (containing 0.1 mM CaCl₂, pH 4.2); 0.1 mM CaCl₂ (pH 4.2) (control). All seedlings were grown in an illuminated incubator at 26 ± 1°C, with a light intensity of 30–50 μmol·m⁻²·s⁻¹. Freshly- treated root tips (1.0 ± 0.1 cm) were collected for the following experiments.

The root elongation from different treatments was determined by measuring the main root length and expressed as relative root elongation (RRE). The RRE was calculated follow the formula: (L_12h-L_0h) × 100%/L_0h· L_12h represented the root length for 12 h treatment, L_0h represented the initial root length before treatment. Cell death of root tips was determined by the method described by Huang et al. (2014a). In brief, root tips (1.0 ± 0.1 cm) were stained with 0.25% (w/v) aqueous solution of Evans blue for 15 min, and then rinsed with distilled water for 30 min. After the stained root tips were immersed in 4 ml N, N-dimethylformamide for 1 h, the solution was collected (containing the dye which had leached from the dead cells) and the absorbance was measured at 600 nm.

Fresh treated root tips (approximately 1 cm) were fixed in 70% osmotically balanced formaldehyde-acetate-alcohol fixative solution buffer for more than 12 h. After root tips were rinsed in ddH₂O three times, dehydrated using an increasing ethanol series (70, 80, 85, 90, 95, and 100%, 1 h for each step), and permeabilized in xylene. The tips were processed and embedded in paraffin wax. The root tips were cut into longitudinal section (10 μm thick) with fully motorized rotary microtome (RM2250, Leica) and adhered to glass microscope slides. Serial tissue sections were deparaffinized in xylene, and rehydrated through a decreasing alcohol series. They were dyed by 1 mg·L⁻¹ DAPI in ddH₂O for 30 s, and observed using a fluorescence microscope (BX53, Olympus, Japan) at 385–400 nm excitation.

Freshly- treated root tips (1.0 ± 0.1 cm) were rinsed in distilled water for 15 min to wash away the Al residue on the surface. After staining with 0.1% (w/v) hematoxylin (containing 0.01% (w/v) KIO₃) for 20 min, the root tips were soaked in distilled water for 10 min, and then observed with a stereo microscope (SZX7, Olympus, Tokyo, Japan).

For determination of total Al content of the root tips, excised root tips (1.0 ± 0.1 cm root segments) were placed in a 1.5 ml eppendorf tube containing 1.0 ml 2 M HCl, as described by Osawa and Matsumoto (2001). The tubes were incubated at 25°C for 24 h, with occasional shaking to release Al from the root tips; the suspending solution was collected and used to determine total root tip Al concentration. CW material was prepared by the method described by Zhong and Lauchli (1993), with minor modifications. Root tips were ground with a mortar and pestle in liquid nitrogen and then homogenized with 7 ml 75% alcohol for 20 min in an ice bath. The sample was then centrifuged at 13,000 × g for 15 min and the supernatant was discarded. The pellets were washed with precooled (4°C) acetone, methanol: chloroform (1:1), and methanol. After drying in an oven at 60°C for 12 h, the pellet was suspended in 1 ml 2 M HCl at room temperature for 24 h with occasional shaking. The Al concentration in the extracts of root tips and CW were determined by inductively-coupled Plasma Optical Emission Spectrometry (ICP-OES; Varian700, Agilent, Santa Clara, California, USA). To clarify the difference of Al enrichment, the percentage of Al content in CWs to total Al in root tips was determined by calculating the ratio of Al content in CWs to the total Al in 1.0 g fresh root tips.

The Al adsorption and desorption kinetics assay was carried out according to the method described by Zheng et al. (2004), with some modifications. A total of 10 mg CW material was placed into a 2 ml column equipped with a filter at the bottom, the adsorption solution (consisted of 100 μM AlCl₃ in 0.1 mM CaCl₂ at pH 4.2) was run through the column using a peristaltic pump set at a speed of 4 mL per 20 min. The adsorption solution was collected at 20 min intervals. The Al adsorbed was desorbed by 0.5 mM CaCl₂ at pH 4.2 at the same speed as adsorption. The accumulative Al adsorbed or desorbed was measured spectrometrically according to Kerven et al. (1989).

NO in peanut root tips was detected by the specific NO fluorescent probe DAF-FM diacetate (Beyotime, Haimen, China) as described previously (Corpas et al., 2009; Yang et al., 2013). After incubation at 25°C for 1 h in the dark with 10 μM DAF-FM diacetate, root tips were washed twice with distilled water. A fluorescence image was taken with a confocal laser scanning microscope (Leica TCS SL, Leica Microsystems, Heidelberg, Germany) at an excitation of 488 nm and emission of 515 nm.

In situ detection of O2·- was performed as described by Dunand et al. (2007) with slight modifications. Root tips were stained for 15 min in a freshly prepared solution of 1 mM nitroblue tetrazolium (NBT) in 20 mM phosphate buffer (pH 6.5). In situ staining of H₂O₂ in root tips was carried out according to the method of Yang et al. (2014) with some modifications. Root tips were incubated in a freshly-prepared solution of 3, 3′-diaminobenzidine (DAB, 1 mg·ml⁻¹) for 4 h. After staining for either ROS, root tips were washed three times with water and observed with a stereomicroscope (Leica DM4000B).

The concentrations of H₂O₂ and O2·- in root tips were measured as described by Sergiev et al. (1997) and Liu et al. (2013), respectively.

The level of lipid peroxidation was assayed by measuring the malondialdehyde (MDA) content, according to the method of Cheng et al. (2015), with some modifications. Approximately 0.2 g root tips were homogenized in 10 ml trichloroacetic acid (TCA) and centrifuged at 13,000 × g for 10 min. An aliquot (2 ml) of the supernatant was mixed with 2 ml 0.6% (w/v) thiobarbituric acid in 10% TCA and heated in a boiling water bath for 15 min. The mixture was quickly cooled in an ice bath and then centrifuged at 13,000 × g for 10 min. The absorbance of the supernatant was determined at 450, 532, and 600 nm. The MDA concentration (C) was estimated using the formula as follows: C (nmol mL⁻¹) = 6.45 × (A532–A600) − 0.56×A450.

Root tips (0.2 g) were homogenized with a mortar and pestle, using 50 mM sodium phosphate buffer (pH7.8), containing 1 mM EDTA, 2% (w/v) PVP, 1 mM PMSF, 1 mM DTT and 0.05% (v/v) Triton X-100. The homogenate was centrifuged at 13,000 × g at 4°C for 15 min and the supernatant was used for determination of enzyme activities. Protein concentration of enzymes extracts was measured by Bradford Protein Assay Kit (Beyotime, Haimen, China), using bovine serum albumin (BSA) as standard.

Lipoxygenase (LOX) activity was determined according to the method of Surrey (1964), with some modifications. The enzyme activity were determined by mixing 50 μl root tip extract with 2.75 ml potassium phosphate buffer (pH 6.5) and 0.2 ml 7.5 mM linoleic acid containing 0.25% (v/v) Tween 20. LOX activity was proportional to the absorbance at 234 nm.

Superoxide dismutase (SOD) activity was measured according to Qureshi et al. (2013) by measuring the inhibition of photochemical reduction of NBT at 560 nm. Catalase (CAT) activity was determined following the method of Hossain et al. (2010) by determining the decomposition of H₂O₂ and monitoring the decrease of absorbance at 240 nm for 3 min. Peroxidase (POD) activity was determined by measuring the rate of guaiacol oxidation and monitoring the absorbance at 470 nm, according to Choudhary (2011). Ascorbate peroxidase (APX) activity was determined according to the method of Asada (1984), measuring the rate of ascorbate oxidation at 290 nm.

PME was extracted according to the method of Rodoni et al. (2010). Root tips (0.2 g) were homogenized with 2 ml 1 M NaCl containing 1% (w/v) polyvinylpolypyrrolidone, and then centrifuged at 13,000 × g for 20 min at 4°C. The supernatant was collected and used for assaying the enzyme activity. The activity was assayed in a mixture containing 1 ml 0.5% (w/v) pectin (pH7.5), 0.4 ml 0.01% (w/v) bromothymol blue (pH7.5), 1.55 ml distilled water (pH7.5) and 50 μl root tip extract. Activity was determined following the method of Hagerman and Austin (1986), measuring the absorbance at 620 nm.

XET activity was detected in situ according to the method of Vissenberg et al. (2000). Root tips were incubated for 1 h in 6.5 μM xyloglucan oligosaccharides (XGO-SRs) dissolved in 2 mM MES buffer. Root tips were washed for 10 min in ethanol: formic acid: water (15:1:4, v/v/v) to remove unreacted XGO-SRs, and then incubated in 5% formic acid to remove apoplastic, non-wall-bound XGO-SRs. These samples were observed by confocal laser scanning microscope (Leica TCS SL, Leica Microsystems) with excitation at 552 nm and emission at 580 nm.

The expression of XTH-32 was analyzed by real-time fluorescent quantitative PCR (qRT-PCR). XTH-32 expression data were presented relative to the expression level of the UBQ10R gene (NCBI accession number: EG030441). RNA was extracted from root tips (0–10 mm) using the Trizol method (Catalog No. 15596-018; Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The cDNA was synthesized from 1 μg total RNA using RevertAid TM first Strand cDNA Synthesis Kit (Catalog No. 1621; Fermentas, Waltham, MA, USA). The primers for XTH-32 were 5′-ATTGAGTTTCTTGGGACTACGTTTG-3′ (forward) and 5′- ATTTGACCATCTCCACTTCCTCT-3′ (reverse). The primers for UBQ10R were 5′-CGCACACTCGCTGACTACAAC-3′ (forward) and 5′-CACGGAGACGGAGGACAAGG-3′ (reverse).

Al significantly inhibited root length of peanut. The inhibition by Al on root elongation of Al-sensitive cultivar ZH2 was greater than that of 99-1507. Compared to the corresponding no-Al control, the root length of ZH2 and 99-1507 under Al stress was 31.9 and 45.0%, respectively (Figure 1A). SNP and cPTIO alone had no effect on root elongation compared to the control. However, co-treatment Al with SNP effectively alleviated the inhibition of root growth induced by Al stress in two peanut genotypes, with the relative root lengths in the Al+SNP treatments being 79.0% (ZH2) and 78.1% (99-1507), compared to the Al treatment alone. But co-treatment Al with cPTIO, a specific NO scavenger, did not significantly change the inhibition of root elongation, compared with Al treatment alone (Figure 1A).

The integrity of the cell membrane during Al stress was evaluated using a non-permeable dye, Evans blue. Compared to the control, SNP and cPTIO alone had no effect on root tip cell death. 100 μM AlCl₃ increased significantly root tip cell death in both cultivars. However, cell death in root tips of ZH2 was 18.8% higher than that of 99-1507 under Al tress. Al-induced cell death was significantly reduced by the addition of SNP, while the addition of cPTIO greatly increased cell death by aggravating the damage to cell membranes (Figure 1B).

To further investigate nuclear changes, the cells were stained with DAPI and examined under a fluorescent microscope after different treatments. The nucleus of normal cell is usually spherical and locates in the middle of the cell without Al stress. Nuclei exposed to Al were flattened, lobed, invaginated or irregular in shape. As shown in Figure 2, NO could prevent PCD caused by Al toxicity. Compared with Al treatment alone, the nuclear remained round after the addition of exogenous NO donor SNP. However, the integrity of cell structure was lost and nuclear condensation presented small crescent-shaped after the addition of NO specific scavenger cPTIO under Al treatment.

Compared with Al treatment alone, NO reduced nuclear deformation, it was verified that exogenous NO inhibited Al-induced PCD in root tip cells of peanut. The effect of cPTIO was in contrast to NO, which confirmed that NO played a negative role in the regulation of PCD induced by Al.

The degree of hematoxylin staining (to visualize Al accumulation) in the root tips of ZH2, exposure to 100 μM Al treatment for 12 h was deeper than that in 99-1507, indicating that Al accumulation was more greater in the Al-sensitive ZH2 (Figure 3A). Compared with Al treatment alone, co-treatment Al with SNP significantly decreased cell staining in root tips of both peanut cultivars, but the effect was more obvious in 99-1507 than in ZH2. Co-treatment Al with cPTIO increased the accumulation of Al in the root tips (Figure 3A).

Al accumulation in peanut root tips and in the root tip CW was detected by ICP-OES. As shown in Figures 3B,C, a significant increase in Al accumulation in root tips and in CWs was observed after Al treatment. Compared with Al treatment alone, co-treatment Al with SNP significantly decreased Al accumulation in root tips and in CWs. On the other hand, co-treatment Al with cPITO significantly increased Al concentration in the root tip CW of ZH2, but not in 99-1507. In the meanwile, there was more Al accumulation in CWs in 99-1507 than in ZH2, and co-treatment Al with SNP decreased the percentage of Al content in CWs to total Al in root tips (Figure 3D). Compared to the control, SNP and cPTIO alone had no effect on the accumulation of Al in the root tips, CW and the percentage of Al content in CWs to total Al in root tips (Figures 3A–D).

Al adsorption and desorption experiments in CWs of two varieties without Al treatment demonstrated that CW of ZH2 had high ability to accumulate Al, it was 25.6% greater in ZH2 than in 99-1507 (Figures 4A,C). Only 65.1% of the total Al absorbed in CW of ZH2 was desorbed within 800 min, whereas the desorption rate of 99-1507 was up to 75.8%, indicating that the ability of CW binding to Al is related to Al tolerance in peanut (Figures 4B,D). After 12 h of Al treatment, the total Al absorbed in the root tip CW of ZH2 and 99-1507 increased by 67.1 and 49.6% respectively, compared to without Al treatment (Figures 4A,C). Compared to Al treatment alone, the addition of SNP decreased 33.8 and 18.5% of the total Al adsorption in CW of ZH2 and 99-1507, respectively, whereas the addition of cPTIO increased the Al adsorption ability in the CWs of ZH2 and 99-1507 (Figures 4A,C). After 12 h of Al treatment, the total Al desorbed in the root tip CW of ZH2 and 99-1507 decreased by 50.8 and 37.3% respectively, compared to without Al treatment (Figures 4B,D). Compared to Al treatment alone, the addition of SNP increased 78.1 and 21.3% of the total Al desorption in CW of ZH2 and 99-1507, respectively, whereas the addition of cPTIO to Al decreased the Al desorption ability in the CWs of ZH2 and 99-1507 (Figures 4B,D).

The production of endogenous NO in peanut root tips was examined using the permeable NO-sensitive dye fluorophore DAF-FM diacetate. The addition of NO donor SNP under no-Al tress showed a strong fluorescence, indicating that SNP could be catalyzed and released NO in peanut root tips (Figure 5A). Compared to the control, a stronger fluorescence was observed in the root tips of ZH2 and 99-1507 following Al treatment. The results showed that Al exposure induced endogenous NO accumulation, and an early burst of NO at 4 h in the root tips of ZH2 (Al-sensitive) and 99-1507 (Al-tolerant), with fluorescence intensity in ZH2 and 99-1507 being 5.4- and 14.1-fold higher than that of the corresponding no-Al treatment (Figures 5A,B). However, with the increase of Al treatment time, the fluorescence in root tips of two cultivars decreased. Co-treatment Al with SNP significantly increased NO level in root tips of the two cultivars, NO production by ZH2 and 99-1507 being 2.3- and 2.0-fold higher, than that of the Al treatment alone, respectively (Figures 5C,D). Co-treatment Al with 50 μM cPTIO effectively inhibited the production of endogenous NO (Figures 5C,D).

The generation of the ROS, O2·- and H₂O₂ in situ in peanut root tips under different treatments was detected histochemically, using NBT and DAB, respectively. Compared with the control, root tips were stained deeply with both NBT and DAB under Al stress (Figures 6A,B). The root tips of ZH2 and 99-1507 stained weakly following the Al+SNP treatment, but stained extensively following the Al+cPTIO treatment. It was found that the generation of both O2·- and H₂O₂ in root tips of the Al-sensitive ZH2 was significantly higher than that of the Al-tolerant 99-1507 under Al stress, indicating higher levels of Al-induced oxidative stress in root tips of ZH2 (Figures 6C,D). Adding SNP significantly decreased the ROS concentration (O2·- and H₂O₂) in both cultivars, but co-treatment Al with cPTIO increased significantly the ROS concentration. SNP alone and cPTIO alone had no significant effect on the ROS accumulation of two cultivars, compared with the control (Figures 6A–D).

The concentration of MDA is an indicator of lipid peroxidation and oxidative damage to membranes, while LOX activity is involved in catalyzing the formation of H₂O₂ derivatives and activating the lipid peroxidation of membranes. The increase in MDA concentration and LOX activity in the Al-stress treatment showed that Al caused severe oxidative damage to plasma membranes in both cultivars. Figure 7A showed that Al treatment caused a significant increase (84.8% higher than the control) in MDA concentration in ZH2 whereas it was only increased by 69.3% in the Al-tolerant 99-1507. Co-treatment Al with SNP clearly inhibited the increase of MDA content induced by Al stress in both cultivars, but the Al+cPTIO treatment increased significantly MDA levels compared to Al alone. LOX activity in ZH2 and 99-1507 increased by 119.8% and 47.5%, respectively, following exposure to 12 h Al stress. Significant differences were observed between LOX activities in the two cultivars at all the treatments, with ZH2 having the higher activities, except for the control. SNP decreased LOX activity, but cPTIO increased LOX activity in both cultivars to above the activities observed in the Al stress treatment (Figure 7B). Compared with the control, SNP alone and cPTIO alone had no significant effect on MDA concentration and LOX activity in two cultivars.

Figure 8 showed that SNP and cPTIO alone had no significant effect on antioxidant enzyme activities in two cultivars. Compared with the control, Al stress increased the constitutive activity of all enzymes, while co-treatment Al with SNP further increased the activities of SOD, POD and CAT, but decreased the activity of APX. Contrary to the effects of SNP, co-treatment Al with cPTIO decreased significantly the activities of SOD, POD, CAT and APX in root tips of both cultivars. The Al-tolerant cultivar 99-1507 exhibited significantly higher levels of SOD, POD, CAT and APX than did the Al-sensitive cultivar ZH2 in both the control and Al-stressed roots (Figure 8).

Al treatment resulted in a significant increase in PME activity in ZH2 and 99-1507 after 12 h of treatment (Figure 9). Compared with Al treatment alone, the PME activity of ZH2 and 99-1507 was reduced significantly by Al+SNP treatment, but was increased significantly under Al+cPTIO treatment (Figure 9). There was no clear difference between the cultivars in terms of PME response. Compared to the control, SNP alone and cPTIO alone had no significant effect on PME activity of two cultivars.

In situ localization of XET activity in peanut root tips was observed using the fluorescent substrate XGO-SRs. The negative control (incubation in MES buffer that lacked XGO-SRs) showed no autofluorescence in response to green light excitation (Figure 10A). Al stress significantly inhibited the XET activity of the two peanut cultivars. The addition of SNP slightly increased XET activity, while the addition of cPTIO significantly increased XET activity under Al stress.

Comparative transcriptomic analysis revealed that the expression of four genes encoding XTH (XTH-1, XTH-2, XTH-23, XTH-32) was significantly changed with prolonged Al exposure times, but the expression level of XTH-32 showed the only statistically significant down-regulation in both cultivars, the other genes showed a transient increase in expression (data not shown). Transcriptional regulation of XTH-32 plays an exclusive role in the expression of XET activity in response to Al stress. Compared with Al treatment alone, the addition of SNP increased the expression of XTH-32 of ZH2 and 99-1507, but the increase was more remarkable in root tips of 99-1507. Exogenous cPTIO application also significantly increased the expression of XTH-32 of ZH2 and 99-1507 (Figure 10B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.