Alleviation of zinc induced oxidative stress by polyamines in Plantago ovata Forsk

Zinc causes toxicity to the plants in an excess concentration and it is manifested by chlorosis, rolling of leaf margins, and disruption of membrane integrity. The heavy metal stress also triggers the stimulation of enzymatic and non-enzymatic antioxidant systems. Polyamines are naturally occurring, secondary metabolites, protecting plants from heavy metal-induced stress. Plants also up-regulate the mRNA expression of Metallothionein in response to heavy metal-induced oxidative stress. The alteration in Metallothionein type 2 (PoMT2) expression of a medicinally important herb Plantago ovata in presence of polyamines like Putrescine, Spermidine, and Spermine in addition to ZnSO 4 .H 2 O by the semi-quantitative and the quantitative methods have been demonstrated in the present study. We have observed reductions in the expression of the Metallothionein type 2 gene in the presence of the aforementioned polyamines which implies their protective and antioxidant properties to ght against the zinc induced stress. 1 mM Put has more in increasing total to Put) by about 36% each in 1000 µM ZnSO 4 treated P. ovata seedlings. Spermidine also enhanced chlorophyll content. 2 mM Put and 0.5 mM Spm have shown even better eciencies in increasing the total antioxidant and DPPH radical scavenging The lipid to Put and Spm supplemented samples by up to about 47% in both cases. Signicant reductions in lipid peroxidation and down-regulation of PoMT2 gene expression indicate the roles of polyamines in partially alleviating Zn-induced oxidative damage.


Introduction
Abiotic stresses are deemed responsible for the reduction in global crop yield which may account for as high as 70% reductions (Liu et al. 2015;Rouphael et al. 2016;Singh et al. 2016). Among the various abiotic stresses, heavy metals are enlisted as the priority pollutants by the United States Environmental Protection Agency (Morkunas et al. 2018). Heavy metals are classi ed as essential and non-essential. Non-essential heavy metals like Pb, Cr, Cd, Hg, and As are toxic even at low concentrations.
Essential heavy metals (Cu, Zn, Fe, etc.), are required by plants for cellular metabolic and physiological processes. However, zinc, like other essential heavy metals, causes severe oxidative stress, senescence and is detrimental in an excessive concentration in plants (Sunitha et al. 2014;Feigl et al. 2015). Heavy metals cause oxidative damage by producing Reactive Oxygen Species (ROS) and free radicals in plants.
In a study made by our group in Plantago ovata, the manifestation of Zn stress is indicated by the stunted growth, chlorosis, reduced chlorophyll, and carotenoid content, and increased lipid peroxidation (Pramanick et al. 2017).
However, plants have adapted various strategies to acclimatize and combat these inclement conditions.
Polyamines are the secondary metabolites produced by all plants in response to oxidative stress which includes heavy metal-induced stress. Polyamines being polycationic compounds, bind to the negatively charged phospholipids, and proteins of the membrane and thus protect them from losing structural integrity (Calzadilla et al. 2014).
The three most abundant polyamines in plants are Putrescine (Put), Spermidine (Spd), and Spermine (Spm). They have speci c functions to perform. Putrescine is known to play a vital role in response to abiotic stress, and maintains the water status of a leaf during drought stress by increasing the proline content (Pál et al. 2015). According to some reports, the levels of Put could be restored only with the exogenous supplementation of Put (Takahashi and Kakehi 2010;Pál et al. 2015). Putrescine aids in stress tolerance by activating the antioxidant system, regulating abscisic acid, and activating Flavonoid synthesis by inducing Phenylalanine ammonia lyase (Takahashi and Kakehi 2010). Spermidine is known to function in plant growth, and development. Mutations of Spermidine synthases result in defective embryo development in the heart stage of Arabidopsis (Imai et al. 2004). Spermine has a varied role in mitigating oxidative stress. It is known to combat the oxidative stress caused due to free radical generation and scavenges the same in the nucleus. Lovaas (1997) reported that polyamines are known to form complexes with metal cations like Cu 2+ , Co 2+ , Zn 2+ , and Ni 2+ and are also able to inhibit metal-catalyzed oxidations or prevent singlet oxygen ( 1 O 2 ) or hydroxyl (·OH) radical formation. The chelation and metal-polyamine-complex formations are directly proportional to the number of nitrogen-groups present in the polyamine and also the polyamine chain length (Lovaas 1997). Lomozik and Wojciechowska (1989) reported that Put forms a seven-membered chelate ring structure with Cu 2+ . This Put-Cu 2+ chelate is not thermodynamically very stable. The higher polyamines like Spm and Spd form metal-complexes much easily than Put given the higher nitrogen content in them. Spm is also known to form a similar seven-membered chelate ring (Lomozik and Wojciechowska, 1989).
The precise role of polyamines in ameliorating heavy metal-induced stress may also be traced back to its function in inducing elevated levels of glutathione (GSH) in plants and subsequent production and accumulation of Phytochelatins both of which are potent metal chelators (Hasanuzzaman et al. 2019). Sengupta et al., (2013) has reported that high doses (50 Gy and 100 Gy) of gamma radiation on imbibed seeds of Vigna radiata resulted in the production of high levels of endogenous polyamine.
When it comes to heavy metal stress in plants, the rst two molecular entities that happen to come in our mind are Metallothioneins and Phytochelatins, because of their metal-chelating and detoxi cation properties (Cobbett and Goldsbrough, 2002). Plants undergo hormesis in response to oxidative stress.
Metallothionein (stress-responsive protein) biosynthesis is one such hormetic activity (Morkunas et al. 2018). It showed up-regulation of its mRNA in response to diverse forms of stresses like exposure to an excess of heavy metals, drought, cold, heat shock, radiation, salinity-among the abiotic stresses (Cobbett and Goldsbrough, 2002;Ghoshal et al. 2013;Moulick et al. 2013).
In the present study, we have determined how plants cope with zinc induced oxidative stress.
Metallothioneins bind heavy-metal ions with mercaptide bonds, while polyamines function in ameliorating the harmful effects of the same in a similar manner. The objective of the present study is to decipher the roles of two important entities-polyamines (Putrescine, Spermidine and Spermine) and Metallothionein type 2 (expression) under zinc stress in Plantago ovata Forsk which has not been reported earlier. P. ovata Forsk is a medicinally and commercially vital herb, cultivated in certain districts of Gujarat and Rajasthan, India. The plant is commonly called psyllium. P. ovata seeds are very interesting because of their husks with huge water-absorbing capacity are used to treat patients with constipation, irritable bowel syndrome, diabetes, and diarrhoea.

Material And Methods
Plant materials, growth conditions and stress treatment: P. ovata seeds (cultivar HI-5) are procured from Madhya Pradesh, India. They are surface sterilized using 10% (v/v) Sodium hypochlorite (NaOCl) for 20 minutes and then washed extensively with autoclaved deionized water to remove traces of chlorine. The sterilized seeds are transferred to autoclaved agarsucrose media (pH-7.6-7.8) composed of 3% sucrose (w/v) [Sisco Research Laboratory (SRL), Mumbai, India], and 0.9% agar-agar (w/v) [SRL, Mumbai, India] contained in plant tissue culture capped glass jars (250 mL). The growth conditions used for maintaining the cultures are-1. Temperature: 22-25ºC, 2.
Relative humidity: 55-60% and 3. Illumination: 1500 Lux for 16 hour duration of photoperiods in a plant tissue culture laboratory. In the case of Zinc Sulphate (ZnSO 4 .7H 2 O) [Merck, India] treatment, 25, 40, and 50 µL solutions of 1M ZnSO 4 .7H 2 O are added to 50 mL of agar-sucrose medium so that the nal concentration comes to 500, 800, and 1000 µM, respectively before sterilization by autoclaving. The lethal dose 50 (LD 50 ) of ZnSO 4 .7H 2 O in P. ovata determined earlier is 1000 µM (Pramanick et al. 2017  The experiments have been designed using 1 and 2 mM Putrescine but only 0.5 mM each of Spermidine and Spermine as higher doses caused shrinkage in P. ovata seedlings.

Preparation of Plant Extracts:
The plant extracts are prepared following the methods of Brolis et al., (1998 ice for about 20 minutes. The ultrasonicated samples are then centrifuged at 10,000 g for 5 minutes.
Then the supernatants are collected and kept at -20°C and further used as 50% ethanolic plant extracts for the determination of total antioxidant content and DPPH radical scavenging activity.

Estimation of Chlorophyll and Carotenoid Contents:
Total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid content are determined following the method developed by Sestak et al. (1971) and Lichtenthaler (1987). 100 mg each of untreated, ZnSO 4 treated and polyamines supplemented shoot and leaf tissues of P. ovata are crushed and homogenized in 5mL of 100% ice-cold acetone [SRL, Mumbai, India] in a mortar. Then the homogenate is centrifuged at 7728 g for 10 minutes at 4 ̊ C in an ultracentrifuge [HERMLE Z383 K, Hermle Machine Company]. Then the clear supernatant is taken in a glass cuvette and spectrophotometric absorbance is measured in the wavelengths of 662, 663, 645, 646, and 470 nm using UV-1800 SHIMADZU UV Spectrophotometer.

Estimation of Total Antioxidant Activity:
To determine the total antioxidant content of the plant extracts of P. ovata, the phosphomolybdenum assay performed by Prieto et al. (1999)  Estimation of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Assay: The DPPH radical scavenging assay is performed by the method followed by Brand-Williams et al., 1995. The reaction mixtures are prepared by adding 0.15 mL of each of the 50% ethanolic extracted samples of P. ovata to 2.85 mL of the working stock of DPPH solution. The reaction mixtures are allowed an hour of incubation in dark at room temperature. Then the spectrophotometric reading of each of the samples is measured at a wavelength (λ max ) of 517 nm.
The percentage radical scavenging activity is measured using the following formula: Where A control is the absorbance of the DPPH reagent without any 50% ethanolic extracted samples and A sample is the absorbance of the DPPH reagent with each of the 50% ethanolic extracted samples.
Estimation of Oxidative damages: The Lipid peroxidation assay is performed following the method of Issawi et al (2018). The amount of malondialdehyde (MDA) produced is indicative of the amount of membrane lipid oxidized. 7 days old P. Reverse Transcription-Polymerase Chain Reaction (RT-PCR): To determine the expression level of MT2 gene in untreated control and ZnSO 4 .7H 2 O treated seedlings (exogenously supplemented with 1 and 2 mM Put, 0.5 mM Spd, and 0.5 mM Spm) of P. ovata, RT-PCR [QIAGEN One-Step RT-PCR kit, QIAGEN, New Delhi, India] is carried out using gene-speci c primers ( Table  2). The RT-PCR reaction mixtures contained 2 µg/50 µL of total RNA. The reaction conditions are shown in Table 3. Table 2 The sequences of the gene-speci c primers of MT2 and β-actin of P. ovata.

Name of gene Forward primer sequence
Reverse primer sequence

Chlorophyll and Carotenoid Content
As depicted in Fig. 1(a), the total chlorophyll contents in 500, 800, and 1000 µM ZnSO 4 treated P. ovata got enhanced by 51.92%, 36.54%, and 2.09% respectively, compared to untreated control seedlings. This increase (though found decreasing with increasing doses of ZnSO 4 ) is evidence of the role of Zn in chlorophyll biosynthesis (Pramanick et al. 2017). 1mM Put supplementation increased the total chlorophyll content in 0, 800, and 1000 µM ZnSO 4 treated seedlings further by 8.2%, 4.48%, and 36.3% respectively, compared to polyamine untreated seedlings of P. ovata. 2mM Put could enhance the total chlorophyll content of 1000 µM ZnSO 4 treated samples only by 10.79%.
With 0.5 mM Spm treatment we observed gradual and statistically signi cant reductions in the total chlorophyll level ( Fig. 1(a)).
Figure 1(b) shows that 0.5 mM Spd has somewhat tried to take up the levels of carotenoid, especially in 500 µM ZnSO 4 treated P. ovata seedlings but, 0.5 mM Spm resulted in signi cant reductions in the carotenoid content of P. ovata ( Fig. 1(b)).
Total Antioxidant Activity 500, 800, and 1000 µM ZnSO 4 treatment showed a dose-dependent increase in the total antioxidant activity in P. ovata. Exogenous supplementation of 1 and 2 mM Put enhanced the total antioxidant contents of (  Fig. 2 depicts that 2 mM Put supplementation has been more effective than 1 mM Put, in increasing the total antioxidant content of the ethanolic plant extracts of P. ovata. 0.5 mM Spd supplementation showed reduced total antioxidant activity of the ethanolic plant extracts in P. ovata. 0.5 mM Spm, when added exogenously to the media, showed an increment in the total antioxidant levels up to 52.49% in 500 µM ZnSO 4 .7H 2 O treated samples compared to polyamine untreated seedlings. On further increasing the dose of ZnSO 4 .7H 2 O, a decrease in the total antioxidant levels is observed (Fig. 2).

DPPH Radical Scavenging Activity
Exogenous addition of 1 mM Put to culture media signi cantly enhanced the DPPH free radical scavenging activity up to (89.14 ± 0.07) % in 1000 µM ZnSO 4 .7H 2 O treated samples.
0.5 mM Spd also enhanced the DPPH radical scavenging activity of 1000 µM ZnSO 4 .7H 2 O treated ethanolic P. ovata extracts to (89.565 ± 0.60) % (Fig. 3). Like 1 mM Put, 0.5 mM Spd was also not potent enough in reducing the rate of membrane lipid peroxidation as depicted in Fig. 4.
However, the exogenous supplementation of 0.5 mM Spm in the media helped in reducing the rate of lipid peroxidation e ciently. It decreased the MDA contents in 0, 500, 800, and 1000 µM Zn treated P. ovata plants by 0.14, 0.45, 0.47, and 0.43 folds, respectively compared to the polyamine untreated seedlings (Fig. 4).
Reverse Put also aided in reducing the MT2 gene expression by 0.44, 0.64, 0.69, and 0.905 folds in Zn untreated and treated P. ovata shoot tissues (Fig. 5). Again, 1mM Put has been more e cient in decreasing the expression of PoMT2.
The exogenously added 0.5 mM Spd also reduced the relative expression of MT2 mRNA in P. ovata by 0.405 fold in 1000 µM ZnSO 4 .7H 2 O treatment (Fig. 5).
The nutrient media supplementation with 0.5 mM Spm, could rather, more e ciently (than Spd) decreased the expression of MT2 mRNA in P. ovata. It reduced the expression of

Statistical Analysis
Results of two-way Analysis of Variance (ANOVA) indicate signi cant changes in the mean values and interactions between the two independent variables ZnSO 4 .7H 2 O and Polyamines concentration (Put, Spd, and Spm) in all the cases of biochemical and molecular biological analyses. At 5% level of signi cance (α = 0.05), we obtained F(cal) > F(0.05) which holds true for all the three polyamines. We, therefore, reject the null hypothesis (which says, there are no signi cant changes in the mean values and that there is no interaction between the two independent variables).

Discussion
Polyamines are often associated with plant growth, DNA and RNA stability, membrane stability, stress resistance, and survival of plant as a whole (Marco et al. 2012;Pál et al. 2015). The effect of exogenous addition of polyamines to alleviate stress depends on various factors like plant species, plant cultivars, duration of given stress, and the dosage of the stress, the tissue type, and also on the way of exogenous polyamine application, that is, through nutrient media supplementation or foliar spray (Soudek et al. 2016).
According to a report Brassica juncea plants exposed to Cd stress showed decrease in the chlorophyll content. Foliar spray of B. juncea with Put enhanced the chlorophyll a content whereas, that with Spd resulted in an increase of both chlorophyll a and chlorophyll b content . Wang et al. (2003) also showed that Hg 2+ and Cr 6+ treatment decreased the chlorophyll content in amaranth leaves which was later reversed by exogenous Spd addition. In the present study, we have also observed that exogenously supplemented Put and Spd could alleviate the effect of ZnSO 4 to some extent and result in an increment of the chlorophyll and carotenoid content in P. ovata.
In a study by Howladar et al., (2018), endogenous Spd and Spm, which also enhanced GSH content conferred Cd tolerance in wheat plants (Howladar et al. 2018). Polyamines can act as antioxidants due to their dual binding properties with anions and cations (Groppa et al. 2007;Cicatelli et al. 2010). Polyamines bind to cations, thereby preventing the formation of ROS (Groppa et al. 2007;Lovaas 1997;Lomozik and Wojciechowska, 1989). As explained by Lovaas, the antioxidant properties of polyamines are attributed by the interaction between the positively charged polyamines and the positively charged metal ions which generate a Coulombic repulsive force and this repulsion is neutralized when negatively charged biomolecules (phospholipids, DNA, and RNA) adsorb polyamines on to them (Lovaas 1997).
Various reports say that polyamines like Put, Spd, and Spm are potent scavengers of free radicals like the superoxide (O 2 ·− ), hydroxyl (·OH), alkoxyl (RO·), peroxyl (ROO·) radicals, singlet oxygen ( 1 O 2 ), and ozone (O 3 ) (Lovaas 1997;Groppa and Benavides 2008;Marco et al. 2012). In this study, we observe an overall increment in the total antioxidant activity with 2 mM Put and 0.5 mM Spm supplementation. Spd, on the other hand, reduced the total antioxidant activity in P. ovata which is in congruence with the results of Mandal et al., (2013) (Mandal et al. 2013). Salvia natans treated with H 2 O 2 showed reduced SOD, GPX, APX, and GR activities with exogenous Put supplementation. Spm also reduced the APOX activity of wheat leaves by 31% but restored the GR activity to the levels of control when added before Cd and Cu (Groppa et al. 2007). According to some reports, exogenous Spm completely restored the GR and SOD activities which were otherwise compromised under Cd 2+ and Cu 2+ treatment in sun ower plants (Chen et al. 2019).
The fact that polyamines act as free radical scavengers is well established. Polyamines and speci cally Spm have the potential to protect DNA from free radical-induced oxidative damage (Groppa et al. 2007;Marco et al. 2012). Therefore, the observation of increased DPPH radical scavenging activity by Put, Spd, and Spm in P. ovata is evident in their function as antioxidants. According to the reports of Sengupta and Raychaudhuri (2017), exogenously added Put signi cantly increased the free radical scavenging activity of gamma-irradiated Vigna radiata seedlings in comparison to Putuntreated seedlings (Sengupta and Raychaudhuri, 2017). Das and Adak, (2015) have found out that 2 mM Spd caused about 24.91% decrease in DPPH radical scavenging activity in Marsilea minuta L. treated with 200 µM CdCl 2 (Das and Adak, 2015). This is again in agreement with our results, where Spd initially showed a decrease in the DPPH radical scavenging activity in P. ovata as well.
Membrane lipid peroxidation is one of the important markers of oxidative stress induced by various abiotic sources. Zn exposure also led to an enhanced rate of lipid peroxidation in P. ovata (Pramanick et al. 2017). Foliar spray of Put and Spd decreased the MDA concentration in Cd treated B. juncea plants . According to the reports of Groppa et al., (2001 and2007) Spm e ciently reduced the TBARS produced during membrane lipid peroxidation in sun ower leaf disk and wheat leaves when exposed to both Cd and Cu (Groppa et al. 2001;Groppa et al. 2007). Putrescine supplementation also reduced the MDA levels in H 2 O 2 treated S. natans signi cantly (Mandal et al. 2013). Cucumber seedlings under hypoxia stress treated with exogenous Spd resulted in decreased malondialdehyde production (Chen et al. 2019). All these results are on par with our observation of reductions in the lipid peroxidation by Put, Spd, and Spm in Zn treated P. ovata seedlings.
This nding of reduced MDA production by Put, Spd, and Spm supplemented P. ovata seedlings can be correlated with the reduced expression of the MT2 gene. The Metallothionein being a stress-responsive gene is induced by heavy metal exposure causing its up-regulation. However, on supplementing the nutrient media with Put, Spd, and Spm we obtained a down-regulation and decreased expression of the PoMT2 gene. Metallothioneins are a group of cysteine-rich, low molecular weight proteins that aid in essential metal homeostasis, detoxi cation of excess heavy metals, and protection from oxidative damage induced by them. They are well-known for their superoxide and hydroxyl radical scavenging activities (Ruttkay-Nedecky et al. 2013). In a previous study by our group, MT2 gene expression studied by qPCR technique showed an enhancement by 4.57 and 5.78 folds in 500 and 800 µM Zn treated P. ovata samples (Pramanick et al. 2017). According to Choudhary et al., (2012), 7 days old radish seedlings pre-treated with 1 mM Spd showed a 3.5 fold decrease in the relative gene expression of RsMT1C (Metallothionein type 1 gene) and is similar to our ndings (Choudhary et al. 2012).
According to Cicatelli et al., (2010), Arbuscular-mycorrhizal-fungal (AMF) association has resulted in upregulation of both MT genes (PaMT1, PaMT2, and PaMT3) as well as polyamine biosynthetic genes ADC and SPDS in Populus alba clone AL35 grown in Cu and Zn polluted soil (Cicatelli et al. 2010). Although a direct relationship between MT2 gene expression and exogenous supplementation of polyamines in nutrient media is not very well elucidated till date, the ndings by Cicatelli et al., (2010) and many more gives an insight into how exogenous polyamines can regulate the expression levels of MT genes in plants and that MT transcriptional expression and action of exogenous polyamines are correlated (Cicatelli et al. 2010). According to Li et al., exogenous Spd supply enhanced MT gene expression in white clover under both well-watered and drought conditions but the same with dicyclohexylamine (DCHA), a secondary amine, resulted in a down-regulation of two leaf-tissue speci c MT gene expression in Trifolium repens (white clover) supplied with 0.05 mM Spd (Li et al. 2016).
Therefore, in this study we hypothesized the ameliorating effects of exogenous Putrescine, Spermidine, and Spermine when applied against oxidative stress induced by ZnSO 4 .7H 2 O in P. ovata. From the results obtained we can infer that the total antioxidant activity, the free radical scavenging activity, and chlorophyll and carotenoid content get enhanced. On the other hand, the simultaneous down-regulations of malondialdehyde production and the Metallothionein Type 2 expression are key symbols of alleviation of Zn induced oxidative stress. We, therefore, propose that excess Zn causes stress which induces the enhanced transcription of the MT2 gene in P. ovata. When confronted with doses of Putrescine, Spermidine, and Spermine against zinc stress, the Metallothionein Type 2 shows a reduced expression due to amelioration of stress by the polyamines.

Declarations
Ethics Approval and Consent to Participate Not applicable

Consent for Publication
Not applicable

Availability of Data and Material
The experiments were performed in replicates and experimental data were generated in our laboratory by us.

Competing Interests
There are no competing interests.

Funding
The authors would like to express their sincere gratitude to the UGC-DAE Consortium for Scienti c Research, Kolkata Centre, West Bengal, India (UGC-DAECSR-KC/CRS/13/RB-01/0808) for nancial support.
Author's Contributions SSRC designed and supervised the research work. PP and SSRC conducted the experiments. AC helped with writing the manuscript. PP and SSRC analyzed the data and wrote the manuscript. All authors read and approved the manuscript. Real Time PCR (qPCR) Analysis and the normalized fold change of MT2 gene in 7 days old P. ovata with increasing doses of ZnSO4.7H2O in the absence and the presence of (a)(i) 1 mM Putrescine, (a)(ii) 2mM Putrescine, (b) 0.5 mM Spermidine, and(c) 0.5 mM Spermine. The data are represented as mean ± standard deviation (SD) (n= 3)