Effect of water-deficit stress on secondary metabolites of Melissa officinalis L.: role of exogenous salicylic acid

[Research]

Effect of water-deficit stress on secondary metabolites ofMelissa officinalis L.: role of exogenous salicylic acid

Jamal Omidi F.¹*, Mohajjel Shoja H.¹, Sariri R.²

1. Department of Plant Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran

2. Department of Biology,Faculty of Science, University of Guilan, Rasht, Iran

ABSTRACT

Melissa officinalis is a perennial herbaceous plant from Lamiaceae family, widely used in food and pharmaceutical industries to add aroma. Drought stress in plants may happen due to the increase in water loss, insufficient water absorption or both. Salicylic acid (SA) is an important secondary metabolite in plants with hormone-like action in some biochemical pathways. Adding it during water-deficit stress, may alter a number of physiological processes, increasing the resistance of plant against possible damage by water loss. The aim of this study was to examine the effect of drought stress (DRU) on secondary metabolites of Melissa officinalis and also the efficacy of salicylic acid treatment to minimize the damages. According to the results, treatment of plant with 0.7 mM salicylic acid had considerable alteration in the quantity of important secondary metabolites. Protein concentration increased in all stages; Chlorophyll a reached to the highest amount; the amount of the MDA increased at the first and second stages in 1/3 fc drought treatment as well as at the third stage in 2/3 fc.  Besides, total phenol content increased at stage I in 1/3 fc drought and the flavonoid content increased in 1/3 fc drought at all stages. Finally, content of anthocyanin was increased at first and third stages 1/3 fc drought, as well as at second stage in 1/3 fc drought. In conclusion, application of salicylic acid (0.7 mM) was useful for improving quality of lemon balm subjected to stress due to water deficit.

INTRODUCTION

Lemon balm, a member of Lamiaceae family, is well known for its medicinal properties (Zargari 1990). It is also used traditionally in many countries, including Iran, to add an aromatic flavor to herbal preparations. In common with many other plants, lemon balm undergoes physiological alterations during a period of drought (Sharafzadeh et al. 2012).

A number of physiological aspects of the plant are altered during water stress. The adverse effect is especially important on photosynthetic capacity and hormonal action. In long term, the growth and productivity of the plant could significantly decrease. However, many plants have specific adaption mechanism to resist many types of environmental stresses. Low water stress causes a significant increase in the concentration of solutes, which promotes the osmotic flow of water out of the plant cells (Douglas 1993). Treatment of the plant during various growth stages with some useful exogenous phytochemicals is a scientific way to adapt various stresses. Salicylic acid (SA) is a secondary metabolite in many plants with a phenolic group in its chemical structure. The compound acts as a phytohormone regulating some physiological processes. It has been reported that binding of SA can alter the activity of some plant proteins (Klessig 2017). It is worth indicating that SA and its derivatives have also multiple protein targets in animals. Some of these proteins, similar to their plant counterparts, are associated with pathological processes. Salicylic acid (SA) alters a number of metabolic pathways including synthesis, oxidation and some biological activities including respiration, photosynthesis and absorption (Borsani et al. 2001). Its use during a low water stress could modify many biochemical pathways leading to improved plant resistance to drought stress (Yazdanpanah et al. 2011). It has been explained that SA is involved in activation of the stress - induced antioxidant system if plants are exposed to stress (Huang et al. 2008). The present study was designed to investigate the effect of salicylic acid, as a plant hormone, to reduce the impact of drought stress on some important characteristics of Melissa officinalis L.

MATERIALS AND METHODS

Materials

The study was conducted in University of Guilan during 2016. A completely randomized program with 3 replications was designed to investigate the effect of salicylic acid on a number of physiological processes of lemon balm (Melissa officinalis L.) under conditions of drought stress. The drought levels of (0, 1/3 field capacity, 2/3 fc) were combined with salicylic acid (0, 0.7 and 1.5 mM) treatments throughout the study period. The media was composed of sand, clay and manure fertilizer with 3:1:1 ratio (Table 1).

Lemon balm seeds were obtained from Iranian Institute of Agricultural Plants, Rasht, Iran. All chemicals and solvents were of analytical grade and purchased from Sigma Chemical Company.

 Table 1. Selected physical and chemical characteristics of the soil.

Abbreviations: P, phosphorous, K, potassium, N, nitrogen and C, carbon.

Methods

Plant growth and harvesting

Seeds were firstly disinfected with formaldehyde (3%, v/v) for 4 minutes followed by washing with distilled water at least 5 times to prevent fungal infection. They were then planted in pots containing 3:1:1 of sand, clay and manure fertilizer, respectively. Harvesting of lemon balm was carried out in three stages, i.e. before (stage I), during (stage II) and after flowering (stage III).

Preparation of samples for scanning electron microscopy (SEM)

The plant material used for histochemical investigation of the length of trichomes, was taken from the fresh samples, as well as the samples preserved in Formalin-aceto-alcohol fixative (FAA) The fixed leaf fragments were dehydrated in increasing alcohol concentration, i.e. 15, 30, 50, 70, 90 and 99.5 %. The plant samples were kept for 15 minutes in each concentration, followed by freeze drying overnight. In the next step, the specimens were placed on the base of an electron microscope (Stub), were gold plated with a Spotter Coated (POLARON SC 7620) and finally, observed using an electron microscope (LEO 1430 VP).

Determination of pigments content

Total chlorophyll concentration is a unifying parameter to determine the effect of any specific interventions. In this investigation, the concentrations of total chlorophyll (Chl a, Chl b) and carotenoids were measured by spectrophotometric method (Lichtenthaler & Wellburn 1983). The chlorophylls a and b and carotenoids were extracted from 0.2 g of leaves using 80% (v/v) aqueous acetone and the mixture was centrifuged. Absorbance of Chl a, Chl b and carotenoids extracts were obtained at 663, 645 and 470 nm, respectively. The concentrations of pigments were calculated as (mg g^-1 fresh weight) from the following relationships:

Chlorophyll a (μg ml^-1) = 12.21 (A663) - 2.81 (A646)            

Chlorophyll b (μg ml^-1) = 20.13 (A646) - 5.03 (A663)

Total Chlorophyll = Chlorophyll a + Chlorophyll b

Carotenoids = [(1000 × OD 470 nm – 1.8 × Chlorophyll a – 85.02 × Chlorophyll b)/198]

Determination of protein content by Bradford method

100 mg of Coomassie Brilliant Blue G-250 dissolved in 50 ml of 95% ethanol and 100 ml of 85% (w/v) phosphoric acid, was added to the mixture. The solution was diluted to 1 liter until the dye was completely dissolved. The resulting mixture was filtered through Whatman#1 paper just before use. A standard curve was obtained by plotting absorbance versus micrograms of protein and used to calculate the concentration of protein considering the dilution factor (Bradford 1976).

Determination of malondialdehyde (MDA) content

Malondialdehyde (MDA) content was measured by the well-known thiobarbituric acid method with a slight modification (Heath & Packer 1968). In practice, 0.5 g of tissue was homogenized in 5 ml of 5% (w/v) trichloroacetic acid and the homogenate was centrifuged at 12000g for 15 minutes at room temperature. An equal volume of thiobarbituric acid (0.5% in 20% trichloroacetic acid w/v) was added to the supernatant and mixed well. The resulting mixture was boiled for 25 minutes at 100° C and centrifuged at 7500g for 5 min. Absorbance of the supernatant was then measured at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The content of MDA was calculated using an extinction coefficient of 155 M^-1cm^-1.

Determination of total phenol

The concentration of phenolics in plant extracts was determined using a spectrophotometric method (McDonald et al. 2001). Practically, a methanolic solution of the extract (concentration 1 mg ml^-1) was used in the analysis procedure. The reaction mixture was prepared by mixing 100 μl of the extract solution, 2 ml of 2% of NaHCO₃, 100 μl of 10% Folin-Ciocalteu’s reagent dissolved in water and, finally, 2.8 ml of distilled water. The samples were incubated in a thermostated bath for 45 min at 45 °Cand the absorbance of solution was obtained at 720 nm. The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained.

A similar procedure was repeated for the standard solution of gallic acid and the calibration line was prepared. Based on the measured absorbance, the concentration of phenolic compounds was read (mg ml^-1) from the calibration line. This content in extracts was expressed in terms of gallic acid equivalent (mg of GA g^-1 of extract).

Determination of flavonoid content

The flavonoids present in the plant extracts were determined using a known spectrophotometric method (Singleton et al. 1999). The examined sample was composed of 1.5 ml of the extract solution with a concentration of 50 μl, and 100 μl in 10 % AlCl₃, 100 μl potassium acetate and 2.8 ml distilled water in 80% methanol. The samples were initially incubated at room temperature for at least one hour and their 415 nm absorbances were then measured. All samples were prepared in triplicate for each analysis and the mean value of absorbance was calculated. The same procedure was repeated for the standard solution of rutin and the calibration line was obtained.

Using this calibration line, the concentration of flavonoids was calculated (mg ml^-1). The content of flavonoids in extracts was, therefore, expressed in terms of rutin equivalent (mg of RU g^-1 of extract).

Determination of anthocyanins

To determine the concentration of anthocyanins, 200 mg of leaf was extracted in 3 ml acidified methanol (methanol: HCl, 99:1, v:v) and stored for overnight in a dark place. The absorbance of the resultant solution was determined at 550 nm and anthocyanin concentration was calculated considering the extinction coefficient of 330 cm^-1mol^-1 (Wagner & Wüthrich 1979).

Variance analysis

The experiment was arranged as a factorial in a completely randomized block design with three replications. Duncan test was employed for grouping of treatment means and variance test for analysis. Statistical analysis was performed using SASS software. Means were compared using Duncan multiple range tests at P ≤ 0.05. The obtained data were analyzed by Microsoft Excel (2010) program.

RESULTS

The scanning electron micrographs from leaf surface at various stages are shown in Fig. 1. The surface was analyzed before treatment at various stages of growth.

The length of trichomes is presented in Fig. 2. The data shows that the length of trichome in stage I, II and III was increased in 1/3 fc drought (35.7, 34.45 and 39.61 mm respectively).

Fig. 1. Scanning electron micrographs of before (stage I), during (stage II) and after (stage III) flowering. The leaf surfaces were prepared using freeze-drying technique, without treatment.

Fig. 2. The effect of salicylic acid (SA) (0, 0.7 and 1.5 mM) treatments, drought stress (DRU) (0, 1/3fc

and 2/3fc) and both factors on trichome length of lemon balm at three stages: before (stage I), during (stage II) and after (stage III) flowering.

The concentrations of photosynthetic pigments (chlorophyll a, b, carotenoids and total chlorophyll) are presented in Table 2. Each column belongs to a specific sampling stage. The highest amount of chlorophyll a in the three stages was related to 0.7 mM salicylic acid (7.71, 9.41 and 10.15 mg g^-1 Fw respectively), that of chlorophyll b in 1.5 mM salicylic acid (6.07, 7.37, and 6.17 mg g^-1 Fw respectively) and that of carotenoids in 0.7 mM salicylic acid

(2.97, 3.27, and 3.59 mg g^-1 Fw respectively). Total concentration of protein is shown in Fig. 3. It is evident from this figure that protein level was increased at I, II and III stages in 0.7 mM treated salicylic acid and 1/3 fc drought samples (2.59, 5.75 and 7.07mg g^-1 Fw respectively). The content of MDA determines the extent of lipid peroxidation. Fig. 4 shows its concentration in the treated samples at various stages.

Table 2. Content of pigments in three stages (I, II, III).

Fig. 3. The effect of salicylic acid (SA) (0, 0.7 and 1.5 mM) treatments, drought stress (DRU) (0, 1/3 fc and 2/3fc) and both factors on total protein content of lemon balm at three stages: before (stage I), during (stage II) and after (stage III) flowering.

It can be observed that the amount of the MDA was increased during first and second stages in 1/3 fc drought treatments (9.70, 10.50 µ mol g^-1respectively) and in third stage of 2/3 fc drought treatment (12.12). On the other hand, as shown in Fig. 5, phenol content was increased in stage I of 0.7 mM salicylic acid and 1/3 fc drought (18.00 mg g^-1 Fw) treated samples as well as in stage II in 1.5 mM salicylic acid and 2/3 fc drought (25.19 mg g^-1 Fw) ones and then in stage III, in 1.5mM salicylic acid and 1/3 fc drought (25.02 mg g^-1 Fw). Fig. 6 represents variations in flavonoid concentration at various stages. It was found that the flavonoid content was increased in 0.7 mM salicylic acid and 1/3 fc drought in all steps (0.50, 0.52 and 0.52 mg g^-1 Fw respectively).

As demonstrated in Fig. 7, the anthocyanin content was increased in the first and third stages in 0.7 mM salicylic acid and 1/3 fc drought (36.95 and 57.93 µmol g^-1 Fw respectively ), as well as in second stage in 1/3 fc drought (40.29 mg g^-1 Fw).

Fig. 4. The effect of salicylic acid (SA) (0, 0.7 and 1.5mM) treatments, drought stress (DRU) (0, 1/3 fc nd 2/3 fc) and both factors on the MDA content of lemon balm at the three stages: before (stage I), during (stage II) and after (stage III) flowering.

Fig. 5. The effect of salicylic acid (SA) (0, 0.7 and 1.5 mM) treatments, drought stress (DRU) (0, 1/3 fc and 2/3 fc) and both factors on total phenol content of lemon balm at three stages: before (stage I), during (stage II) and after (stage III) flowering.

Fig. 6. The effect of salicylic acid (SA) (0, 0.7 and 1.5mM) treatments, drought stress (DRU) (0, 1/3 fc and 2/3 fc) and both factors on total flavonoid content of lemon balm at three stages: before (stage I), during (stage II) and after (stage III) flowering.

Fig. 7. The effect of salicylic acid (SA) (0, 0.7 and 1.5 mM) treatments, drought stress (DRU) (0, 1/3 fc and 2/3 fc) and both factors on total anthocyanin content of lemon balm at three stages: before (stage I), during (stage II) and after (stage III) flowering.