Reactive oxygen species scavenging mechanisms associated with polyethylene glycol mediated osmotic stress tolerance in Chinese potato

Influence of polyethylene glycol (PEG) mediated osmotic stress on reactive oxygen species (ROS) scavenging machinery of Chinese potato (Solenostemon rotundifolius (Poir.) J. K. Morton) was investigated. Five genotypes of Chinese potato were raised in Murashige and Skoog (MS) basal medium containing 6–benzylaminopurine (BAP, 1 mg L–1) along with various concentrations of PEG–6000 mediated stress conditions (0, –0.2 and –0.5 MPa) and evaluated for osmotic stress tolerance in vitro. The medium containing PEG–6000 had a detrimental effect on plantlet growth and development while compared with the control. Accumulation of H2O2 was lower in Sreedhara and Subala and higher in Nidhi under PEG stress, which was evident by in situ detection in leaves. Lipid peroxidation product such as malondialdehyde (MDA) content was increased due to PEG stress which was more in susceptible genotype than that in tolerant ones. An enhanced ROS–scavenging antioxidant enzyme was observed under stress with respect to the control. The enzymes of ascorbate–glutathione cycle showed an important role in scavenging ROS. The imposition of PEG stress also increased the non–enzymatic antioxidants viz., the ascorbate and reduced glutathione content which was prominent in tolerant genotypes in comparison to susceptible. The present study indicated that, Sreedhara and Subala showed more tolerance to osmotic stress with better ROS scavenging machineries which would be the lines of interest for augmenting future breeding strategies in this climate resilient minor tuber crop.

Explants, growth conditions, treatments and growth parameters. Nodal explants (5-6 mm) of the five genotypes of Chinese potato were collected from poly house and pre-treated with fungicide (Carbendazim 0.15%) for 10 min. The explants were cleaned thoroughly in running tap water, surface sterilized in 2% sodium hypochlorite solution for 3 min. and rinsed thrice in sterile water. Culture medium included Murashige and Skoog 11 basal medium with 6-benzylaminopurine (BAP, 1 mg L -1 ) and sucrose (30 g L -1 ) solidified with phytagel (3 g L -1 ). PEG-6000 at a concentration of 118.0 and 197.0 g L -1 was incorporated in MS medium to induce osmotic stress of -0.2 and -0.5 MPa, respectively and pH was adjusted to 5.8 ± 1. Equal amount of culture media (15 ml) was poured into the test tubes (25 × 100 mm, Borosil, India) and sterilized in a steam autoclave (Remi, India) at 105 kPa for 15 min. Nodal explants of all the five genotypes were inoculated in the MS medium with different levels of PEG. The explants inoculated in MS + BAP alone were maintained as the control. All cultures were incubated at 25 ± 2 °C with 16 h photoperiod and 40 µmol m -2 s -1 fluorescence light (Phillips, India) for 6 weeks. The experiment was set up in a 5×3 factorial completely randomized design (fCRD) replicated thrice with triplicate determinations. Shoot and root proliferation of in vitro plantlets were observed at 6 weeks of inoculation.

Estimation of H 2 o 2 , in situ detection of H 2 o 2 and lipid peroxidation. H 2 O 2 content in leaf tissues
of Chinese potato under different treatments was estimated following the method of Velikova et al. 12 by measuring the oxidation product at 390 nm. H 2 O 2 was estimated using standard concentrations of 0-100 µM and was expressed as µM g -1 FW. H 2 O 2 produces a brown stain in plant tissues which is localized by 3,3-diaminobenzidine (DAB) 13 . Vacuum infiltration of whole leaves was done with DAB solution (1 mg mL -1 ) followed by incubation at dark for 12-16 hrs with mild agitation (80-100 rpm) in a rotating shaker (Tarson, India). The chlorophyll was completely removed from the leaves by treating with ethanol:acetic acid:glycerol (3:1:1) and incubated in the circulating water bath (Thermo Fisher Scientific, USA) at 75 °C. The DAB polymerized reddish-brown spots produced on site of the leaf tissues was photographed by a 10 mega prixel digital camera (Sony, India).
The cellular damage caused by ROS can be estimated by lipid peroxidation of the cell membrane; which is measured in terms of malondialdehyde (MDA) content 14 . MDA content (nM g -1 FW) in the leaves was determined using the extinction coefficient 15 of 155 mM -1 cm -1 .
SOD activities (EC 1.15.1.1) was estimated by measuring the inhibition ability of the nitroblue tetrazolium chloride (NBT) reactions 16 . The unit of SOD enzyme inhibiting 50% NBT was expressed as U g -1 FW of leaf sample. Similarly, CAT activity (EC 1.11.1.6) was determined as the rate of H 2 O 2 scavenged which was measured by decrease in absorbance at 240 nm. Quantification of CAT activity was performed by its molar extinction coefficient (40 mM -1 cm -1 ) following the method of Aebi 17 .
Determination of non-enzymatic antioxidants: ascorbate (ASA) and reduced glutathione (GSH) content. Ascorbate content (ASA) was estimated following a standard curve plotted with known concentrations of ascorbate in leaf tissue and expressed as mg g -1 FW 23 . GSH was estimated following standard method 24 and quantified using standard concentrations of reduced glutathione (µmol g -1 FW).
Statistical analysis. Data on in vitro growth parameters, biochemical estimations and ROS scavenging antioxidants were recorded at six weeks of inoculation under control and PEG treatments (-0.2 and -0.5 MPa). The experiments were conducted in 5 × 3 factorial completely randomized design (fCRD). Analysis of variance (ANOVA) 25 was performed to test the significance at probability level P ≤ 0.05 and P ≤ 0.01. Tukey's test was performed to compare significant differences among the mean values 26 . Graphical representations are mean of three replications with triplicate determinations.

Results
Influence of PEG mediated osmotic stress on in vitro growth responses of Chinese potato. Analysis of variance for in vitro growth parameters (Table 1) showed significant effects across the genotypes and PEG treatments. Growth of in vitro plantlets decreased significantly under PEG treatments (-0.2 and -0.5 MPa) while compared with the stress-free control. Days to bud break was severely delayed in response  www.nature.com/scientificreports www.nature.com/scientificreports/ to PEG stress (Fig. 1a). After 6 weeks of culture, decrease in number of shoots, leaves, and roots, length of shoots and roots were more pronounced in the higher osmotic treatment (-0.5 MPa) ( Fig. 1b-f, P ≤ 0.01). Sreedhara and Subala demonstrated less variation in growth parameters than other genotypes under moisture stress as compared to control.
Influence of PEG stress on H 2 o 2 production, in situ detection and lipid peroxidation of Chinese potato. ANOVA for H 2 O 2 accumulation and lipid peroxidation (MDA content) revealed significant variation under PEG induced osmotic stress, genotypes and genotype x PEG interactions at P ≤ 0.01 (Table 2). H 2 O 2 activity in leaf tissues of Chinese potato was increased with increase in PEG stress (Fig. 2a). Figure 2b represents lipid peroxidation (MDA content) in leaf tissues of control and PEG treated Chinese potato. A significant increment in MDA content was observed among the studied genotypes; which was more in Nidhi than Sreedhara and Subala (Fig. 2b). Figure 3 depicts the in situ detection of ROS through DAB staining in Chinese potato leaves. The PEG treated leaves turned to dark brown which was more pronounced in Nidhi than in Sreedhara and Subala. As expected, the control leaves showed no brown precipitation (Fig. 3).   Table 2). The activity of all seven antioxidative enzymes increased in Chinese potato leaves when exposed to varying concentrations of PEG stress (Fig. 4a-c). The SOD activity was increased steeply in genotypes Sreedhara and Subala under stress conditions as compared to control (Fig. 4a). Followed by this, the CAT activity showed the similar increasing trend with the increased osmotic stress level (Fig. 4b). The magnitude of increase in CAT activity was highest in Sreedhara (68.50%), as compared to Nidhi (29.25%) under higher stress conditions over control. The activity of GPX also significantly increased upon PEG stress (Fig. 4c), being more pronounced in Sreedhara and Subala compared to Nidhi and CO-1.
Influence of PEG stress on ASA-GSH cycle of Chinese potato. The enzymes of ASA-GSH cycle also showed an increment in their activities when imposed to PEG mediated osmotic stress (Fig. 5a-d). Subala registered significantly higher induction of all four enzymes of this cycle viz., APX, MDAR, DHAR and GR; whereas, Nidhi showed the least induction in the similar conditions.
Following PEG imposed stress in leaves, ascorbate (Fig. 5e) and reduced glutathione (Fig. 5f) was increased significantly in all the tested genotypes with respect to their control. Ascorbate content under osmotic stress (-0.5 MPa) was increased to the tune of 83.04% in Subala followed by 60.08% in Sreedhara; the same was lower in Nidhi in all the treatments while compared with other genotypes (Fig. 5e). In a similar way, reduced glutathione was increased by 1.63 and 1.14-fold in Subala and Sreedhara, respectively at higher PEG stress as compared to their controls (Fig. 5f).
Overall result implied that the genotypes Sreedhara and Subala performed better under induced PEG stress with the lower accumulation of H 2 O 2 and MDA which further impaired by induction of various antioxidants (enzymatic and non-enzymatic) as the powerful scavengers of ROS. TVM and CO-1 showed moderately susceptible and Nidhi was characterized as susceptible to moisture stress.

Discussion
We have taken an in vitro approach to study the effect of PEG mediated osmotic stress on ROS-scavenging mechanisms among five genotypes of Chinese potato. In vitro evaluation is an efficient and quick approach for understanding stress tolerance which showed the similar effect as compared to the labour-intensive field-based screening 27 . So far, there is no report available on effects of PEG mediated osmotic stress on growth responses of Chinese potato in vitro. PEG at high molecular weight acts as a non-intrusive osmotic agent, that overcast the water potential in the culture media and thereby, widely used in osmotic stress tolerance studies in plants 28 . Growth responses of nodal explants of Chinese potato significantly decreased when PEG was incorporated in MS medium. The first detrimental effect of PEG in the shoot multiplication media was evident by a gradual delay in bud break from lower to higher stress as compared to the control. The bud break was earlier by 10 days of inoculation in Sreedhara and Subala, whereas, it was delayed by 3 weeks in Nidhi at -0.5 MPa stress. The overall growth in terms of number of leaves, number of shoots, length of shoots and rooting was highly affected due to PEG imposed media as compared to the control. Under PEG stress condition, Subala and Sreedhara maintained the growth by registering up to 20% decrease in shoot proliferation and rooting, which reflects its inherent tolerance towards osmotic stress. On the contrary, Nidhi showed an indication of susceptibility towards PEG stress by exhibiting more growth retardation in vitro. Our previous studies in taro, a carbohydrate-rich tuber crop, demonstrated the detrimental effect of PEG on shoot proliferation in vitro 29 . Reports have also shown that limited www.nature.com/scientificreports www.nature.com/scientificreports/ water availability restricted cell division and elongation and thereby hinders plant growth and development 30,31 . The extent of plant growth inhibition depends on the genotype and the severity of stress; which was prominent in our study across the studied genotypes.
ROS generated inside the cell as a result of oxidative stress can target the lipid membranes resulting in elicitation of the lipid peroxidation 31,32 . Increased MDA content is considered as a hallmark of membrane lipid peroxidation and also an indicator of free radical prevalence in the tissues. To understand whether PEG mediated osmotic stress results in the induction of ROS in Chinese potato, we have measured the level of H 2 O 2 and MDA content in control and stressed leaves. Our results have shown a significant increment in both H 2 O 2 and MDA content; which shows the involvement of oxidative stress incurred by imposing to PEG stress among the studied genotypes. Sreedhara and Subala registered lower H 2 O 2 and MDA accumulation (up to 20%) at higher PEG (-0.5 MPa) as a sign of lower osmotic imbalance. However, Nidhi possessed higher osmotic imbalance with higher accumulation of H 2 O 2 and MDA (up to 85%) than its control. In support of our results, Fu et al. 33 have shown that MDA content of cassava exhibited a linear change under higher PEG treatments. Studies have shown that MDA content and H 2 O 2 accumulation was about two to four-fold in susceptible genotypes than the tolerant ones [34][35][36] . From our studies, it was evident that Sreedhara and Subala maintained lower H 2 O 2 and MDA content irrespective of PEG doses which reflect more membrane stability and also an indication of early defense to combat osmotic stress imposed by PEG.
To further confirm, we have also carried out in situ detection of H 2 O 2 in leaves by DAB staining. H 2 O 2 along with peroxidases (a haem-containing protein) oxidizes DAB, which is visualized by a dark brown precipitation in the leaves. Among the genotypes, Nidhi followed by CO-1 showed extensive dark stained DAB sites in the leaves making the leaves turning completely black which is correlated with higher accumulation of H 2 O 2 compared to Subala and Sreedhara (where only leaf edges were stained). Chakraborty and Pradhan 35 documented higher accumulation of H 2 O 2 was associated with the appearance of darkly stained DAB spots in leaves of susceptible varieties of wheat than the tolerant ones; which is in support of our results. Oxidative burst in leaf tissues through www.nature.com/scientificreports www.nature.com/scientificreports/ formations of free radicals due to PEG stress was characterized by increased ROS production as reflected by a sharp increase in H 2 O 2 production and also, increased level of MDA. This holds true in the in situ DAB test for ROS generation; where the more extensive brown stain was detected in Nidhi leaves than that of Subala and Sreedhara.
The ability of the fast and effective antioxidant response in stress situations can reflect the stress tolerance ability of the plant 37 . In our study, activities of all antioxidative enzymes were significantly increased under osmotic stress across the Chinese potato genotypes which were higher in Subala and Sreedhara as compared to other genotypes. Khanna-Chopra and Selote 38 reported a higher level of antioxidative machineries in stressed plants over non-stressed plants. Similarly, Wang et al. 39 reported that the osmotic adjustment ability in tolerant cultivar is more efficient over susceptible ones. SOD is the first line of antioxidant which rapidly scavenges O 2 and restricts the production of OH 40 . We have observed upto 52.6% increase in the activity of SOD in Sreedhara followed by Subala (45.6%) over control, suggesting that these genotypes have inherent O 2 .radical scavenging ability than other studied genotypes (Nidhi, 15.24%).
H 2 O 2 produced is further reduced to H 2 O by two major antioxidative enzymes -CAT and GPX. CAT activity increased with increase in PEG stress and was found to be 1.7-fold induced in Sreedhara at higher stress conditions; showing its efficiency in reducing the toxic effects of generated H 2 O 2 . Our previous study also showed an increase in CAT activity under PEG induced osmotic stress in taro 29 . We have also observed a significant increment in the activity of H 2 O 2 scavenging enzyme GPX in Sreedhara (upto 7.8-fold change) at higher stress. This can be correlated with in situ DAB staining data; where very less brown stain was observed in Sreedhara leaves restricted at the edges only. This enhanced activity could explain the detoxification of H 2 O 2 mainly occurred through GPX than CAT. Therefore, from our results it is apparent that GPX plays a stringent role in H 2 O 2 elimination than CAT in Chinese potato. The genotype Sreedhara performed better by registering maximum fold change in these enzyme activities. Previous report showed enhanced activity of GPX was greatly associated with prolonged drought spell in wheat varieties 35 .
Our results also showed that ascorbate-glutathione cycle (ASA-GSH) played an important role on scavenging the over production of H 2 O 2 due to osmotic stress in Chinese potato genotypes. APX, GR, MDAR and DHAR involved in ASA-GSH cycle were also higher in stressed plants than that in control 39,41 . This series of enzymatic and non-enzymatic antioxidants such as SOD, CAT, GPX and DHAR delays leaf senescence induced by PEG stress 42 .
ASA acts as an important defensive antioxidant against overproduction of ROS induced by oxidative stress 43,44 . Resistant varieties maintained higher ascorbate content over susceptible ones under different levels of drought www.nature.com/scientificreports www.nature.com/scientificreports/ stress 39 . This antioxidant plays a major role either in preventing or in lowering the risk caused by ROS in higher plants; by removal of H 2 O 2 via ASA-GSH cycle. Reduced glutathione also participated in ROS inhibition and induced drought stress in Arabidopsis plants 45 . In our study, the magnitude of increase in these two antioxidants was lower in Nidhi than Subala and Sreedhara; showing the inability of Nidhi to cope up with induced oxidative stress and making it as susceptible.

Conclusions
In vitro PEG mediated osmotic stress resulted in oxidative burst as evident by accumulation of H 2 O 2 and lipid peroxidation. Increment in enzymatic and non-enzymatic antioxidants under stress implied a positive hallmark of stress tolerance in the present study. Enhancement in the accumulation of ascorbic acid and reduced glutathione accompanied by increased GR activity depicts the involvement of ASA-GSH cycle in osmotic stress tolerance in Chinese potato. Of the five genotypes tested, Sreedhara and Subala exhibited distinct antioxidative mechanisms for scavenging of ROS and protecting the plants from deleterious effects of PEG mediated osmotic stress. Considering all the above data, our study revealed that Sreedhara and Subala were tolerant, TVM and CO-1 were moderately tolerant and Nidhi was susceptible to PEG stress. The detailed information on ROS scavenging machineries would be useful for intensification of breeding strategies in Chinese potato for development of tolerant lines to harsh environments.