Nitric oxide acts as an antioxidant and inhibits programmed cell death induced by aluminum in the root tips of peanut (Arachis hypogaea L.)

Aluminum (Al) causes programmed cell death (PCD) in plants. Our previous studies have confirmed that nitric oxide (NO) inhibits Al-induced PCD in the root tips of peanut. However, the mechanism by which NO inhibits Al-induced PCD is unclear. Here the effects of NO on mitochondrial reactive oxygen species (ROS), malondialdehyde (MDA), activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), expression of alternative oxidase (AhAOX) and cytochrome oxidase (AhCOX) were investigated in peanut (Arachis hypogaea L.) root tips treated with Al. The results showed that Al stress induced rapid accumulation of H2O2 and MDA and increased the ratio of SOD/APX. The up-regulation of AhAOX and AhCOX expressions was not enough to inhibit PCD occurrence. Sodium nitroprusside (SNP, a NO donor) decreased the ratio of SOD/APX and eliminated excess H2O2 and MDA, thereby inhibiting Al-induced PCD in the root tips of peanut. The expression of AhAOX and AhCOX was significantly enhanced in Al-induced PCD treated with SNP. But cPTIO (a NO specific scavenger) supply had the opposite effect. Taken together, these results suggested that lipid peroxidation induced by higher levels of H2O2 was an important cause of Al-induced PCD. NO-mediated inhibition of Al-induced PCD was related to a significant elimination of H2O2 accumulation by decreasing the ratio of SOD/APX and up-regulating the expression of AhAOX and AhCOX.

As a major factor, aluminum (Al) toxicity limits crop productivity in acid soil. Al inhibits root elongation growth and disrupts the uptake of nutrient and water in plants. Al enhanced the production of reactive oxygen species (ROS), leading to mitochondrial respiration inhibition and ATP depletion in plant cells 1 . Al exclusion and tolerance mechanisms are associated with mitochondrial metabolism, especially organic acid transport and mitochondrial activity 2 . Programmed cell death (PCD) is a process of cellular suicide controlled by genes. Al toxicity may be the result of Al-induced PCD in the root tips of barley 3 . The negative regulation of PCD alleviated Al toxicity in yeast 4 . Our previous researches showed that Al induced mitochondria-dependent PCD in Al-sensitive peanut cultivar rapidly 5 .
Nitric oxide (NO) has been considered as a signal regulator involved in plant growth and stress tolerance. Al stress changes the homeostasis of endogenous NO in plants 6 . Exogenous NO donor sodium nitroprusside (SNP) treatment can alleviate Al toxicity by ameliorating effectively Al-induced mitochondrial respiratory dysfunction in wheat root tips 7 . The former studies of our group had showed that NO suppresses PCD induced by Al in peanut root tips 8 . NO may be an antioxidant to postpone PCD in barley aleurone layers 9 . However, the mechanism by which NO inhibits Al-induced PCD is unclear.
As an important power station, mitochondrion is mainly responsible for electron transport, oxidative phosphorylation, and energy metabolism in animal and plant cells. Higher concentrations of Al treatment opened the mitochondrial membrane permeability transition pore (MPTP) and released cytochrome c (Cyt c) into the cytoplasm, thereby induced PCD in peanut root tips 10 . In Arabidopsis thaliana, Al toxicity induced mitochondria-dependent PCD 11 . Oxidative stress increased ROS generation, the opening of MPTP, and activation of proteases, leading to the occurrence of PCD in Arabidopsis cells 12 . In the early stages of PCD in Results evaluation of mitochondrial function. To evaluate the mitochondrial function, the membrane potential and Cyt c value were detected. The mitochondrial membrane potential can be expressed as the fluorescence intensity of the fluorescent probe Rh-123. The results showed that the fluorescence intensity of the control and Al treatment were 0.89 and 0.56, respectively. After Al treatment, Cyt c value decreased from 21.4 nmol·μg −1 Pro to 9.2 nmol·μg −1 Pro. This indicated that the mitochondrial function was pure, integral, and viable.

Effects of Al on mitochondrial O 2
.− , H 2 o 2 , and lipid peroxidant. When plants are exposed to adversity stress, a lot of ROS are produced and excess ROS do harm to plants. To study the effects of Al on mitochondrial ROS and lipid peroxidant, peanut seedlings were exposed to 100 μmol·L −1 AlCl 3 solution at different times. As Al treatment time extended, O 2 .− production rate increased sharply to the peak at 8 h and then decreased slowly (Fig. 1A). Compared to the control, the mitochondrial O 2 .− production rate reached a peak in the root tips of peanut at 8 h of Al treatment. As shown in Fig. 1B, the mitochondrial H 2 O 2 content of peanut root tips was increased after Al treatment. Al treatment for 1 h increased significantly H 2 O 2 content and then kept up at a higher level compared with the control. MDA content is always used as an indicator to estimate the level of lipid peroxidation. The results showed that the MDA content was increased sharply as Al treatment time increased (Fig. 1C). Compared with the control, MDA content of mitochondria was increased by 2.2 fold at the time of 4 h Al treatment in the root tips of peanut. At 12 h and 24 h, MDA content increased by 3.6 fold and 6.3 fold, respectively. MDA content was increased significantly in a time-dependent manner.

Effects of Al on mitochondrial antioxidase activities.
To study the effects of Al on mitochondrial antioxidase activities, the activities of SOD and APX were measured. The activity of SOD was gradually decreased as Al treatment time prolonged ( Fig. 2A). Compared with the control, SOD activity of peanut root tips was decreased by 32.1% at 4 h Al treatment. At 12 h and 24 h, SOD activity had dropped by 60.7% and 71.4%, respectively. The activity of APX was sharply decreased as Al treatment time increased (Fig. 2B). Compared to the control, the activity of APX in the root tips of peanut was reduced by 50% at 4 h of Al treatment. At 12 h and 24 h, APX activity had dropped by 87.5% and 95.3%, respectively. As Al treatment time increased, the ratio of SOD/ APX was rapidly increased (Fig. 2C).
Relationship between root cell death and H 2 o 2 , MDA, SOD/APX in peanut root tips. As shown in Fig. 3A,C, H 2 O 2 content in mitochondria of the peanut root tips was significantly positively correlated with not only cell death (R 2 = 0.993) but also SOD/APX (R 2 = 0.885). However, the correlation between cell death and superoxide was very poor (R 2 = 0.044). There was a significantly positive relationship between lipid peroxidation and cell death in peanut root tips (R 2 = 0.935) (Fig. 3B). Fig. 3D showed that SOD/APX was significantly positively correlated with cell death in the root tips of peanut (R 2 = 0.857).

Effects of NO on O 2
.− , H 2 o 2 , and MDA in the root tips of peanut. To study the effects of NO on mitochondrial ROS and lipid peroxidation, peanut seedlings were exposed to different chemical treatments. As shown in Fig. 4A, NO decreased mitochondrial O 2 .− production rates in the root tips of peanut. Compared with Al treatment alone, SNP effectively inhibited the production of mitochondrial O 2 .− , while cPTIO (a NO specific scavenger) significantly increased the production rate of O 2 .− . As shown in Fig. 4B, NO inhibited H 2 O 2 production by mitochondria in the root tips of peanut. Compared with Al treatment, SNP significantly inhibited the production of mitochondrial H 2 O 2 , while cPTIO intensified H 2 O 2 content. As shown in Fig. 4C, NO decreased mitochondrial MDA content in peanut root tips. Compared to Al treatment alone, SNP significantly reduced mitochondrial MDA content in the root tips of peanut, while cPTIO significantly increased MDA content.

Effects of NO on SOD, APX, and SOD/APX in the root tips of peanut.
To study the effects of NO on mitochondrial antioxidase activities, the activities of SOD and APX were measured under different chemical treatments. As shown in Fig. 5A, Al treatment inhibited the activity of mitochondrial SOD in the root tips of peanut. Compared to Al treatment alone, SNP significantly increased the activity of SOD, while cPTIO significantly www.nature.com/scientificreports www.nature.com/scientificreports/ reduced the activity of SOD. As shown in Fig. 5B, Al treatment inhibited the activity of mitochondrial APX in the root tips of peanut. Compared to Al treatment alone, SNP significantly increased the activity of APX, while cPTIO significantly reduced the activity of APX. The addition of SNP decreased the ratio of SOD/APX, which was increased by Al treatment, while cPTIO supplement increased the ratio of SOD/APX (Fig. 5C).

Effects of NO on AhAOX and AhCOX expression during Al-induced PCD. The expression of AhAOX
was rapidly increased and then slowly decreased under Al stress (Fig. 6A). After 1 h of Al treatment, the expression of AhAOX was increased, indicating that Al boosted the expression of this gene. At 4 h Al treatment, the expression of this gene was the highest, which was 10.3 times than that of the control. The expression of AhAOX was then reduced. The expression of this gene at 12 h Al treatment was 2.22 times of the control. As shown in Fig. 6B, compared to Al treatment alone, the addition of SNP increased significantly the expression of AhAOX, an increase of 919.65%; cPTIO supplement decreased the expression of AhAOX, but there was no significant difference compared with Al treatment alone.
The expression of AhCOX was rapidly increased and then slowly decreased under Al treatment (Fig. 6C). After 1 h of Al treatment, AhCOX expression was increased, indicating that Al motivated the expression of this gene. At 4 h Al treatment, the expression of this gene was the highest, which was 17.92 times than that of the control. Then the expression of AhCOX was gradually decreased. The expression of this gene at 12 h was 1.30 times than that of the control. As shown in Fig. 6D, compared with Al treatment alone, the addition of SNP significantly increased the expression of AhCOX, an increase of 237.92%, while the expression of AhCOX for cPTIO supplement was significantly higher than that of Al treatment.  . 1), SOD, APX, SOD/APX (Fig. 2), AhAOX (Fig. 6A), AhCOX (Fig. 6C), and cell death in the root tips of peanut with different Al treatment time, hierarchical cluster was conducted to analyze the interaction between NO and ROS on peanut response to Al stress. The results indicated that Al stress inhibited www.nature.com/scientificreports www.nature.com/scientificreports/ the activities of SOD and APX (Fig. 7a-A), increased the ratio of SOD and APX, up-regulated the expression of AhAOX and AhCOX (Fig. 7a-B), promoted the accumulation of H 2 O 2 , O 2 .− (Fig. 7a-C), and MDA, resulting in Al-induced PCD in peanut root tips (Fig. 7a-D). Under Al stress, the physiological parameters are clustered well to four groups (A-D). There is a causal relationship between group A and D. Group B and C are paralleled.

Hierarchical cluster analysis of ROS in NO inhibiting Al-induced PCD in the root tips of peanut.
Based on the data of mitochondrial O 2 .− , H 2 O 2 , MDA (Fig. 4), SOD, APX, SOD/APX (Fig. 5), AhAOX (Fig. 6B), AhCOX (Fig. 6D), and cell death in the root tips of peanut with different treatments, hierarchical cluster was used to analyze the interaction between NO and ROS on peanut response to Al stress. The results indicated that NO donor SNP promoted the activities of SOD and APX (Fig. 7b-A), decreased the ratio of SOD and APX, up-regulated the expression of AhAOX and AhCOX (Fig. 7b-B), reduced the accumulation of H 2 O 2 and O 2 .− ( Fig. 7b-C), leading to inhibition of Al-induced PCD in the root tips of peanut. cPTIO supply had the opposite effects. During NO inhibiting Al-induced PCD, the physiological parameters are clustered well to three groups (A-C). There is causal relationship between group A and C. Group B and C are paralleled. Group C is also divided into two subgroups, which have a more direct causal relationship. www.nature.com/scientificreports www.nature.com/scientificreports/ with Al-induced PCD in the root tips of peanut is H 2 O 2 rather than O 2 .− (Fig. 3A). Cadmium (Cd) induced the accumulation of H 2 O 2 in the roots of Pinus sylvestris L., induced xylem formation and accelerated senescence 19 . Low concentrations of Al stimulated PLC and PLD signaling pathways to lead to ROS production, followed by the caspase-like protease to execute cell death 20 . Moreover, the results of correlation analysis indicated that lipid peroxidation induced by higher levels of H 2 O 2 might be an important cause of Al-induced PCD (Fig. 3A,B). NO can combine with O 2 .− to form peroxynitrite (ONOO − ), which can induce cell death. Because O 2 .− was not major ROS during Al-induced PCD, in fact, ONOO − was rarely generated.

Discussion
Nitric oxide mediates inhibition of Al-induced PCD by decreasing the ratio of SOD/APX to scavenge excess H 2 o 2 . The decrease of SOD/APX ratio contributed to the elimination of H 2 O 2 (Fig. 3C), so the increase of SOD/APX ratio may be associated with Al-induced PCD in peanut roots (Fig. 3D). NO donor SNP enhances the antioxidant capacity of wheat seedlings under Al stress 21 . NR-dependent NO production alleviated Al-induced oxidative stress in the roots of red bean 22 . NO suppressed Cassia tora root sensitivity to Al by inactivating the cell wall peroxidase activity and reducing H 2 O 2 production 23 . As an antioxidant, NO increased the activities of SOD and CAT, delay PCD in barley aleurone layers 9  www.nature.com/scientificreports www.nature.com/scientificreports/ is crosstalk and synergistic action between NO and H 2 O 2 24 . NO treatment reversed Al-induced reactive oxygen species toxicities by promoting the expression of antioxidant enzymes 25 . The decrease of NO level promoted the accumulation of ROS and induced the expression of pathogen-related proteins (PRs) to protect cells from Cd toxicity 26 . NO and ROS can induce cell death alone or synergistically 27 . In the present study, Al stress decreased SOD and APX activity and raised membrane lipid peroxidation in peanut apex, while NO activated antioxidant enzymes (SOD, APX) system to protect the peanut root tip from ROS damage. The result is consistent with the findings of Wang and Yang 28 . Because the decreasing range of APX activity was larger than that of SOD activity, excessive H 2 O 2 could not be removed in time. With the prolonging of Al treatment time, Al stress increased H 2 O 2 production and MDA accumulation, which was related to the rise of SOD/APX ratio. The linear relationship between SOD/APX and H 2 O 2 content also clearly indicated that the content of H 2 O 2 increased with the rise of SOD/APX ratio (Fig. 3C). Acute stress generated ROS and reactive nitrogen species (RNS) and lead to APX degradation. The regulation of APX mediated by NO may be a redox sensor of oxidative stress 29 . H 2 O 2 alleviated salt-induced oxidative stress by modulating APX and SOD activities in cotton 30 . Similar to the result of Fan et al. 31 , NO up-regulated the activities of SOD and APX. But the rising range of APX activity was larger than that of SOD activity, resulting in the decrease of SOD/APX ratio. By decreasing the ratio of SOD/APX, SNP eliminated excess H 2 O 2 and decreased MDA, thereby inhibiting Al-induced PCD in the root tips of peanut. But cPTIO supply had the opposite effect. www.nature.com/scientificreports www.nature.com/scientificreports/ an important part in plant physiology 32 . AOX can effectively reduce the production of mitochondrial ROS in plant cells and decrease cell injury 33 . The lack of mitochondrial AOX increased susceptibility to PCD in transgenic plants, but induction of mitochondrial AOX prevented PCD by down-regulating the cytochrome pathway 34,35 . Salicylic acid and H 2 O 2 treatment up-regulated the expression of AOX in wild type tobacco, reduced ROS accumulation in mitochondria and delayed PCD occurrence 36 . AOX acts as a buffer that determines the threshold of PCD induction 37,38 . The expression of AOX was significantly up-regulated in tobacco suspension cells treated with 500 μmol·L −1 AlCl 3 . Overexpression of AOX could enhance the tolerance of tobacco suspension cells to Al stress 11 . The PCD degree of tobacco with AOX knockout was more serious 27 .

Inhibition of Al-induced PCD by NO is related to the enhancement of
As the center enzyme of complex IV in the electron transport chain, COX is related to the mitochondrial respiratory metabolism and ATP synthesis 39 . In the present study, the results showed that Al stress raised membrane lipid peroxidation and up-regulated the expression of AhAOX and AhCOX, which was not enough to inhibit PCD occurrence. The expression of AhAOX and AhCOX presented periodic fluctuation within 24 hours, speculating that they might be related to the biological clock. However, NO can enhance significantly the expression of AhAOX and AhCOX, protect the peanut root tip from ROS damage. As for how NO regulates their expression via a biological clock, it needs further study.
Determination of root cell death. Fresh roots were stained with 0.25% (w/v) Evans blue solution for 15 min. After washing with deionized water for 10 min, ten root tips (10 mm) were excised and digested or 1 h in 4 ml N,N-dimethylformamide at room temperature. The absorbance of Evans blue was measured at 600 nm 40 . www.nature.com/scientificreports www.nature.com/scientificreports/ Isolation of mitochondria from the root tips of peanut. According to Panda's method 37 , mitochondria were separated from peanut root tips. After rinsing with distilled water, 5 ml mitochondrial extract buffer (0.3 mo1·L −1 mannitol, 25 mmol·L −1 MOPS-KOH (pH 7.8), 10 mmol·L −1 tricine, 8 mmol·L −1 cysteine, 1 mmol·L −1 EGTA, 0.1% (w/v) BSA, and 1% (w/v) PVP-40) were used to homogenized about 3 g fresh treated root tips on ice-bath. After 15 min centrifugation at 1 500 × g, the homogenate supernatant was centrifuged at 14 000 × g for 15 min. The precipitate was washed for 3 times by using mitochondrial suspension buffer (0.4 mo1·L −1 mannitol, 1 mmol·L −1 EGTA, 10 mmol·L −1 tricine, pH 7.2). The final pellet was resuspended with mitochondrial suspension buffer of appropriate volume. To detect the viability of mitochondria, a suspension stained with 0.02% Janus Green B was observed under oil microscope. According to the methods of Braidot et al. 41 and Zhang 42 , the www.nature.com/scientificreports www.nature.com/scientificreports/ membrane potential and Cyt c value were detected, respectively. The method of Bradford 43 was used to determine protein concentration, which represented the mitochondrial concentration.
Assay of mitochondrial enzyme activities. The activity of mitochondrial SOD was determined by nitroblue tetrazolium (NBT) display method 44 . The total volume of 3 mL mixture included 1.5 mL of 0.05 mol·L −1 phosphate buffer (pH 7.8), 0.3 mL of 130 mmol·L −1 Met, 0.3 mL of 750 μmol·L −1 NBT, 0.3 mL of 100 μmol·L −1 EDTA-Na 2 , 0.3 mL of 20 μmol·L −1 riboflavin, 0.05 mL mitochondria extract, and 0.25 mL distilled water, respectively. 4 to 6 tubers were used as control, which the enzyme solution was replaced with buffer. After blending, two control tubes were placed in the dark, and other tubes under 4000 Lx fluorescent lamp were reacted for 20 min (the consistent light situation was required, the reaction time shortened at high temperature but the reaction time extension at low temperature). After the reaction ended, taking the control tube treated with dark as blank, OD 560 of other tube were determined respectively. 50% suppression of NBT photoredox reaction was one enzyme activity unit. SOD activity = 2 × [OD 560 (control) − OD 560 (sample tube)] × volume of sample tube (mL)/ OD 560 (control) × Weight × volume of solution determined (mL). The unit of enzyme activity was U·μg −1 Pro. The activity of mitochondrial APX was determined as follows 45 . 3 mL reaction mixture contained 50 mmol·L −1 K 2 HPO 4 -KH 2 PO 4 buffer (pH 7.0), 0.1 mmol·L −1 EDTA-Na 2 , 0.3 mmol·L −1 AsA, and 0.1 mL mitochondrial extract. After adding H 2 O 2 , the absorbance changes within 10 to 30 s at 290 nm were immediately determined at 20 °C and the reduction of AsA and enzyme activity in unit time were calculated. The unit of enzyme activity was μmol ASA·mg −1 Pro·min −1 .

Detection of o 2
.− production rate and H 2 o 2 content. The production rate of superoxide anion free radical (O 2 .− ) was determined according to the method of Zhan et al. 15 . 0.5 mL of mitochondrial extract was put into a test tube, respectively. Then 0.5 mL of 50 mmol·L −1 phosphate buffer (pH 7.8) and 1 mL of 1 mol·L −1 hydroxylamine hydrochloride were added successively. After mixing, the mixture was kept for 1 h at room temperature. After 1 mL 17 mmol·L −1 aminobenzene sulfonic acid and 1 mL 7 mmol·L −1 alpha naphthylamine were added, mixing and displaying for 20 min at room temperature. With 0.5 mL phosphate buffer as a blank control, the absorbance at 530 nm was determined. ] was used to express the production rate of O 2 .− by stoichiometry. Nitrite content was calculated by the standard curve. According to the method of Sergiev 46 , H 2 O 2 content was measured. 1 mL of mitochondrial extract, 2 mL of 1 mol·L −1 KI, and 1 mL of 0.1 mol·L −1 phosphate buffer (pH 7.0) were added to the test tube. According to a H 2 O 2 standard curve, the absorbance at 390 nm measured after 20 min shake was converted into H 2 O 2 concentrations.
Determination of mitochondrial MDA content. 0.2 mL mitochondrial extract (distilled water as a control) was put into a test tube and was added by 1 mL 0.6% thiobarbituric acid (TBA). Then it was bathed in boiling water for 15 min. After cooling, the homogenate was centrifuged at 1500 × g for 10 min to measure the absorbance at 532 nm, 600 nm, and 450 nm, respectively. Malondialdehyde (MDA) content was calculated according to the method of Zhan et al. 15 . .− and H 2 O 2 ) contents and oxidative damage, which resulted in PCD occurrence. NO partially prevented Al-induced decay of activities of SOD and APX, enhanced the expression of AhAOX and AhCOX, then reduced ROS production, which inhibited the production of PCD. The solid line represents Al 3+ effect. The dashed line represents the inhibitory effect of NO on cell death. The sharp head represents promotion, whereas the flat head represents suppression.