Influence of exogenous polyamines on antioxidant defence and essential oil production in valerian ( Valeriana officinalis L . ) plants under drought stress

The objective of this study was to determine the effects of foliar application of polyamines (PAs) on antioxidant defence and essential oil production of valerian (Valeriana officinalis L.) grown under different drought stress treatments (100, 70, 50 and 30% available water content). This study was carried out using pots in greenhouse condition. Drought–stressed valerian seedlings were sprayed with 1 mM concentration of each putrescine (Put), spermidine (Spd) and spermine (Spm). The results showed that drought stress significantly affected most biochemical characteristics of valerian plants. Characteristics including leaf relative water content, chlorophyll a and b contents were decreased, while carotenoids and electrolyte leakage were increased with the increase of water stress. In this research, defensive characteristics comprising proline content, soluble sugars, catalase, and ascorbate peroxidase were increased followed by drought stress to ameliorate the adverse effect of it. Results revealed that foliar application of Spd and Spm provoked the antioxidant enzymes activity as well as proline accumulation in valerian which alleviate the membrane damages. Consequently, Spd and Spm increased photosynthetic pigments which act as energy supply for plant growth and production. Here, putrescine had detrimental effects on CAT activity and Chl a content. Albeit, PAs presented remarkable effects under moderate drought stress condition but it showed reverse trends in severe drought stress condition. In terms of quantity and quality yield, drought stress reduced root growth but increased the concentration of essential oils. PAs are able to alleviate water deficit-induced diminish root growth. These results suggest that in moderate drought stress, growers can use PAs to increase productivity valerian.


INTRODUCTION
Valerian (Valeriana officinalis L.) which belongs to the family Valerianaceae is an important medicinal plant that is, due to its mild sedative and sleep aid, widely used in traditional medicine (Bos et al., 1996).The subterranean organs of valerian contain several compounds including the essential oils and their sesquiterpenoid derivatives (valerenic acids), epoxy iridoid esters (valepotriates) and their decomposition products such as baldrinal and homobaldrinal, and alkaloids (Isetts, 2010).Valerian is the 8th top-selling herbal supplement in North America (Blumenthal, 2001), making it very attractive to improve agronomical practices and in general its production increases.Therefore, eliminating the unfavourable circumstance conditions for valerian is one of the primary and most important strategies in it`s production.The genus Valeriana contains about 200 species, which are mostly found in the cold and temperate regions and majority of species possessed high water requirement (Bernatb, 1999).It is reported that the lower water saturation levels had an adverse effect on valerian production (Berbec, 1965).Considering literature, plant growth and functionality suppression under drought stress condition result from morphological, physiological and molecular changes.In limiting water states, plants tend to close their stomata, resulting in reduction of CO 2 flow into the leaves for fixation.As result, far less amounts of NADPH + H + and ATP are consumed within the Calvin cycle.Consequently, the concentration of NADP + , and thus that of potential electron acceptors for electron transport chain decreases.Thereafter, electron flow on free oxygen and finally oxygen radicals are induced.The ROS are highly reactive and can seriously damage plants by protein degradation, DNA fragmentation, lipid peroxidation, membrane destruction, and ultimately cause cell death (Beligni and Lamattina 1999).
In plants, several defence systems contribute with together to alleviate effects of any stress factor.One of the important defensive systems is the involvement of phytohormons (Farooq et al., 2009).Literatures have emerged that abiotic stress modulated the accumulation of polyamines in many plant species (Alcázar et al., 2011;Shi and Chan, 2014), and these observations revealed the possible implication of polyamines to increase plant stress tolerance.Polyamines (PAs) including putrescine (Put), spermidine (Spd), and spermine (Spm), are mainly accumulated in plants exposed to environmental stresses, and play a key role in stress tolerance which depends on species and stress intensity and duration (Tavladoraki et al., 2012).Limited water resources beside progressive demand for food production encouraged researchers to improve water use in agriculture.Recently, researches pursue to apply polyamines exogenously to ameliorate the adverse effect of environmental stresses such as drought stress (Mustafavi et al., 2015).For instance, Nayyar et al., (2005) found that exogenous application of Put and Spd substantially improved the drought tolerance in soybean.It was also shown that plants mainly change range of essential oils production as defense system when exposed to stress conditions (Ramakrishna and Ravishankar, 2011).Saeidnejad et al., (2013) reported that drought stress increased essential oil content of Bunium persicum (Boiss.)B. Fedtsch.plants.Recently researches have deliberately applied drought stress during the cultivation of medicinal plants in order to stimulate the accumulation of pharmaceutical products such as essential oils.In this regard there is a fundamental question which exogenously applied substance can trigger in plant the accumulation of secondary metabolites with no impact on medical use.Therefore, in this study we followed the hypothesis that by providing polyamines exogenously, the synthesis of pharmaceutical metabolites would be increased.
This study was aimed to determine the effects of several drought stress states accompanied with exogenous application of PAs on biochemical characteristics and essential oil production of valerian plants.

MATERIALS AND METHODS
A pot experiment was conducted in greenhouse condition at the University of Maragheh, Maragheh, Iran.Pots with 8 L content were filled with 12 kg of soil (clay: clean sand; 2/1 v/v).The soil had been passed through a mesh number 10.Then, 20 days-old valerian seedlings were obtained from medicinal plants institute, Tehran, Iran.Uniform seedlings were collected and then transplanted into each pot.The pots were placed under a rain-shelter based on completely randomized design.During the next 30 days after transplantation, plants were irrigated with tap water, and then divided into four lots subjected to different water levels.Drought stress levels were measured using control plants with 100 % water availability (L1), 70 % (L2), 50 % (L3) and 30 % (L4) of available water.The pots were weighed daily and water was added to maintain soil moisture content.In this project, polyamines including putrescine, spermidine and spermine, (Sigma Chemical Co.) were sprayed 2 times in 1 mM concentration.In order to eliminate the effect of water from test results, untreated plants were sprayed with distilled water.The first spray was made 35 days after transplanting and the second repeated after 15-days.The plants were sprayed with a manual pressure pump at an average of 10 ml.All of the observations, except essential oil content, were taken three days after later foliar application of PAs.
Water status of leaves was determined by measuring relative water content (RWC).The leaves were subsequently rehydrated in distilled water for 4 h to obtain the turgid mass (TM), and dried for 48 h at 72 •C to obtain the dry mass (DM).RWC was calculated by the ratio: RWC = [(FM − DM)/(TM − DM)] × 100.For measurement of electrolyte leakage, 20 leaf discs from the young fully expanded leaves were placed in 50 mL glass vials, rinsed with distilled water to remove electrolytes released during leaf disc excision.Vials were then filled with 30 mL of distilled water and allowed to stand in the dark for 24 h at room temperature.Electrical conductivity (EC 1 ) of the bathing solution was determined at the end of incubation period.Vials were heated in a temperature-controlled water bath at 95°C for 20 min, and then cooled to room temperature and the electrical conductivity (EC 2 ) was measured.Electrolyte leakage was calculated as percentage of EC 1 /EC 2 .
Chlorophyll-a, -b, and carotenoid were determined according to the method of Arnon (1949).Fresh leaves were taken from the plants and triturated in 80 % acetone.The absorbance of the extracts was measured at 663, 642 and 472 nm using a spectrophotometer (BioTek, PowerWave, USA).Proline content of leaf tissues was estimated spectrophotometrically following the ninhydrin method described by Bates et al., (1973).Total soluble sugars were extracted and determined by the anthrone method of Riazi et al., (1985).
The shoot tissues (0.5 g fresh mass) were homogenized in 2 mL of 100 mM potassium phosphate buffer, pH 7 containing 1 mM of EDTA and 1 % (w/v) polyvinylpyrrolidone (PVP).The extract was then centrifuged at 4°C for 15 min at 12.000 ×g in a cooled centrifuge.This supernatant was used to measure the activities of guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT).Activity of GPX was determined at 25°C with guaiacol (Bergmeyer, 1974).Activity of APX was measured by following the rate of hydrogen peroxide-dependent oxidation of ascorbic acid (Nakano and Asada 1981).Finally, the activity of CAT was assayed following H 2 O 2 decomposition.
Finally, 50 g of dry root was taken for determination of essential oil percentage using Clevenger instrument (European Pharmacopoeia, 2005).Essential oil yield was determined using the following formula: Essential oil yield = Essential oil percentage × Root yield.Analysis of variance appropriate to the experimental design was conducted using SPSS software (version 16).Means of each trait were compared according to Duncan multiple range test at P<0.05 by means of MSTATC software.

Relative water content (RWC) and electrolyte leakage
Regarding Cell membrane stability is considered to be one of the best physiological indicators of drought stress tolerance.Electrolyte leakage (El) level increased from 20 to 42 %, when AWC dropped from 100 to 30 %. Except for Put, plants treated with PAs showed a significant reduction of stress-induced electrolyte leakage depending on its type and drought stress level (Table 1).Under moderate drought stress (70 % AWC), application of Spd and/or Spm vigorously decreased El but with increasing the water shortage their positive effect was markedly reduced (Figure 1a).Similar results were also reported by Shaddad et al., (2011) who reported that at very severe drought stress condition, application of polyamines did not alleviate the adverse effect of drought stress.
Results reported in the present paper showed that exogenous Spd and Spm could effectively alleviate membrane damage induced by water shortage up to 50 % AWC (Figure 1).The observation is in harmony with the results of Chattopadhayay et al., (2002) on rice and Li et al., (2014) on white clover plants.They proposed that exogenous spermidine and spermine effectively protected cells against damage by inhibition of protease and RNAse activities, which probably helped in maintaining membrane integrity.In addition, polyamines may mediate a decrease in ion fluxes across the vacuolar membrane by blocking fast-activating vacuolar channels under salt stress, as suggested by Pottosin et al. (2014).Besford et al., (1993) reported that dicotyledons mainly accumulate Spd and Spm with a decline in Put.Therefore, according to their findings it seems that Put had detrimental effect on dicotyledons plant such as valerian.
Figure1: Effect of foliar PAs treatments on valerian leaves (a) electrolyte leakage and (b) proline concentration under different available water content levels (AWC).

Osmotic adjustment
Proline is considered to act as a compatible osmoticum and is perhaps involved in drought tolerance (Bandurska, 2000).In the present study, in response to drought stress, proline concentration was gradually increased reaching a peak value at 50 % AWC and then commenced to reduce (Table 1).The lowest proline content was obtained with normal irrigation (control), while the highest was obtained at irrigation with 50 % AWC.Proline accumulation is in accordance with the fact that many higher plants accumulate free proline in response to drought stress (Farooq et al., 2009).Several possible roles have been attributed to accumulation of proline: osmoregulation, detoxification of free radicals, conservation of nitrogen and energy for the post-stress period and regulating the stress protective proteins (Khedr et al., 2003).Although stressed plants sprayed with Spd and Spm exhibited 19.7 and 7.2 % higher accumulation of proline compared to PA-untreated shoots, their effects mainly depend on soil AWC (Table 1).In this respect, Spd and/or Spm treatments stimulated proline accumulation whenever soil AWC lowered and reached to 50 % (Figure 1b).Our results are in line with the results of Kubis et al., (2014).They concluded that cucumber seedlings treated with PAs exhibited a definitely higher stress-evoked proline accumulation.Shi and Chan (2014) reported that polyamine and proline biosynthesis pathways might share some common substrates, and exogenous application of polyamines might result in more substrates for proline biosynthesis, especially under stress conditions.
Total soluble sugars concentration increased with increasing the drought stress levels up to 50 % AWC, however, differences were not significant, and then they reduced markedly.Foliar application of PAs failed to revert changes in carbohydrates concentration caused by the water shortage treatments (Table 1).In general, the inability of PAs in reducing water loss (Table 1) suggests that osmotic adjustment in valerian leaves cannot be regulated by PAs under drought conditions.

Antioxidant enzymes activity
Results indicated that drought stress had a significant effect (P < 0.01) on antioxidant enzymes activity in leaves of valerian plants.The activities of catalase (CAT) and ascorbate peroxidase (APX) were increased with the increase of drought stress from the control (100 % AWC) to 50 % AWC.However, further increase in water shortage reduced CAT activity, but maximum value of APX activity was achieved at 30 % AWC.In contrast to CAT and APX, the activity of guaiacol peroxidase (GPX) tended to decrease by increased water deficit.Maximum GPX (10.7 units/min .mg.pro) contents were noted from wellwatered valerian plants, which were decreased significantly upon exposure to drought stress (Table 1).Liu et al., (2011) reported that catalase activity was higher under mild drought stress than under severe drought stress and well-watered treatments.The increase of CAT and APX activity in plants under drought stress was also reported in other studies (Anjum et al., 2012;Huseynova, 2012).
In current study, except for Put, exogenous PAs significantly promoted activities of CAT relative to untreated plants.High and low CAT activities were recorded from 1 mM Spd and Put, respectively (Table 1).Efficacy of PAs application on APX and GPX activities depends on water stress levels.According to Figure 2a, under moderate drought stress, only Put induced marked changes in APX activity, but by further increase in drought stress levels, treatments had not statistically significant differences with control, and adverse effects of drought stress eclipsed PAs effects.In the case of GPX activity, at all drought stress levels, exogenously applied Put and/or Spd had no significant effect on GPX activity, while Spm had dual effects, under moderate and severe drought stress, positive and negative, respectively (Figure 2b).The same result was obtained in study of Li et al., (2014) who showed that exogenous Spd significantly promoted activities of antioxidant enzymes under drought stress.These findings revealed that enzymatic antioxidant activities of valerian leaves were substantially induced by Spd and Spm application (Table 1).Enhanced antioxidant defense system in PAs treated plants resulted in improving cell membrane stability, as demonstrated by lower electrolyte leakage (Table 1).Pottosin and Shabala (2014) reported that polyamines may play a dual role in the ROS scavenging process.First, PA may play a critical role in drought stress signalling to confer adaptive responses (Toumi et al., 2010).On the other hand, PAs are known to significantly enhance activity of both enzymatic (Radhakrishnan and Lee, 2013) and non-enzymatic (Asthir et al., 2012) antioxidants.Therefore, the PA control over the balance between ROS production and scavenging may "shape" H 2 O 2 signal, conferring differential stress responses between species and genotypes.

Photosynthetic pigments
Drought stress significantly affected chlorophyll a, b, and carotenoids contents (Table 1).The results revealed that Chl."a, b" contents decreased by decreasing the soil moisture content.Drought stress up to 70 % AWC induced a slight on the chl-a, chl-b of valerian leaf, then it reduced them markedly by the further increase in the level of drought stress.Contrary to these results, plants carotenoid values increased by drought stress increasing up to 50 % and then commenced to reduce (Table 1).Our results about chl-a and chl-b changes in response to drought stress are in agreement with reports of Liu et al., (2011).
Reduction in chlorophyll pigments concentration can be as a drought response mechanism in order to minimize the light absorption by chloroplasts (Pastenes et al., 2005).Since carotenoid plays an important role in photo-protection (Munne-Bosch and Penuelas, 2003), the increased carotenoid content under drought conditions, indicate a higher need of photo-protection by carotenoid (Elsheery and Cao, 2008).
PAs foliar application had significant effect on chlorophyll a and b contents depending on available water content levels.According to Figure 3a, with increasing water shortage up to 50 % AWC, exogenously applied PAs had significant effects on chl.a content, but under severe conditions, differences among the treatments were not statistically significant.Under moderate drought stress, Spd and Spm application increased chl.a content, while Put had detrimental effect.In the case of chl.b content, application of Spd had positive effect when sprayed on stressed-plants.At all water shortage levels, Spm foliar application slightly increased chl.b content (Figure 3b).In normal plants, moderate drought stress lead to increasing in chla/b ratio while this ratio tends to decrease followed by severity of drought stress (Figure 3c).Results (Figure 3c) depicted that foliar application of PAs did not make any remarkable changes on chla/b ratio.Our findings manifested that Spd could positively influenced total chlorophyll (a + b) content comparing to other PAs in all drought stress states (Figure 3d).All PAs treatments had not significant effect on carotenoid content (Table 1).In connection with these results, Chattopadhayay et al., (2002) found that the exogenous application of polyamines enhanced the total chlorophyll level of salt-stressed rice plants.This enhancement effect of Spd and Spm may be attributed to increased stability of thylakoid membranes (Chattopadhayay et al., 2002).Thus polyamines could bind to the negatively charged phospholipid head groups on membranes, thereby influencing stability and permeability characteristics of these membranes.

Growth and Essential oil production
Plant aerial biomass as well as root biomass was severely inhibited under drought stress, and both of them decreased with increasing the water shortage (Table 2).Aerial parts growth decreased by 28-50 % while subterranean organs growth decreased by 21-35 %.Therefore, roots were less affected by drought stress than aerial parts (Table 2).Root is the first organ to come into contact with water stress in the rhizosphere, thus supplying assimilate for this organ may be a best strategy for their resistance.Application of PAs significantly improved root and above ground organs growth under drought stress (Table 2).Maximum root biomass (economical part) was recorded from Spm foliar application, which was at par with Spd treatment (Table 2).PAs exogenously applied to plants grown under drought stress mitigated the adverse effect of stress on plant growth which may be attributed to stimulatory effects of PAs on defence systems and chlorophyll protection as as our data indicated (Table 1).Our results are in line with previous studies (Shaddad et al., 2011;Amooaghaie, 2011) where exogenous PAs alleviate stress-induced growth inhibition possibly due to protection of membranes and minimization of oxidative damage.
Essential oil percentage increased when soil AWC dropped to 50 %, and then it declined with drought stress intensifying (Table 1).

Figure 2 :
Figure 2: Effect of foliar PAs treatments on Ascorbate peroxidase (APX) activity (a) and Guaiacol peroxidase (GPX) activity (b) in leaves of valerian under different available water content levels (AWC)

Figure 3 :
Figure 3: Effect of foliar PAs treatments on chlorophyll a content (a), chlorophyll b content (b), chlorophyll a/b ratio (c) and total chlorophyll (a + b) content in leaves of valerian under different available water content levels (AWC).

Table 1 :
Effect of foliar PAs treatments on some physiological and biochemical traits of valerian under drought

Table 2 :
Effect of foliar PAs treatments on quantitative and qualitative yield of valerian under drought stress To sum up, present study suggest that foliar application of Spd and Spm could ameliorate the deleterious effects of drought stress by stimulating the antioxidant enzymes activity and increasing the accumulation of proline, which protect cell membrane integrity and decrease chlorophyll loses.Albeit, PAs possessed less tangible effects on valerian plants in severe drought stress state but, it is more effective in moderate drought stress state through induction of biochemical changes.It is inferred that PAs which is economical and environment friendly alternative can be implicated to improve productivity of valerian in current scenario of drought and climate change.Plant Signaling and Behavior, 6: 243-250.Doi: 10.4161/psb.6.2.14317 Amooaghaie R. 2011.Role of Polyamines in The Tolerance of Soybean to Water Deficit Stress.World Academy of Science, Engineering and Technology, 56: 498-502.Anjum S.A., Farooq M., Xie X.Y., Liu X.J., Ijaz M.F.2012.Antioxidant defense system and proline accumulation enables hot pepper to perform better under drought.Scientia Horticulture, 140: 66-73.Doi: 10.1016/j.scienta.2012.03.028Arnon D.I. 1949.Copper enzymes in isolated chloroplasts.Polyphenoloxidase in Beta vulgaris.