Mitigation of drought stress in pot marigold (Calendula officinalis) plant by foliar application of methanol

Introduction

Calendula officinalis L. (marigold) from the Asteraceae family is an annual herbaceous plant that has been used for herbal medicine, pharmaceutical industry and chemical composition. Flowers of this plant contain compounds that can be broadly applied as an antiseptic, anti-inflammatory and cicatrizing as well as a light antibacterial and antiviral agent (Khalid et al. 2010). Changes in physiological and photochemical processes due to a change in environmental conditions such as water deficiency or agricultural practices lead to a change in plant growth and productivity (Pallardy 2010).

Water deficiency imposes one of the most important constraints to photosynthesis, plant growth and crop productivity (Hosseinzadeh et al. 2015). The impact of the drought on plant species depends on the variety, severity and duration of the stress as well as on the development stage (Simova-Stoilova et al. 2008). Closed stomata which reduce transpiration and conserve water in plants is the first mechanism of plants to face dehydration stress (Sikder et al. 2015), which in turn limits carbonfixation, reduces NADP⁺ regeneration by the Calvin Cycle and decrease the photochemical activities (Arora et al. 2002; Monakhova and Chernyadev 2002; Barbosa et al. 2015). Exposure of plants to stress is known to induce the formation of reactive oxygen species (ROS), which are involved not only in damage mechanisms but also in cell growth processes (Bernstein et al. 2010). ROS such as superoxide, hydrogen peroxide and hydroxyl radical are highly reactive and can seriously disrupt normal metabolism through oxidative damage on lipids, proteins and nucleic acids (Ashraf 2009). Also, water deficit can damage pigments and plastids, reduce chlorophyll a, chlorophyll b and other carotenoids, hydrolyze proteins and prevalent photochemical reactions in most plants (Reddy et al. 2004). Recent investigations have shown that chlorophyll and its derivatives act as antioxidants to prevent oxidative DNA damage and lipid peroxidation both by chelating reactive ions and by scavenging free radicals (Hsu et al. 2013). As a result, the induction of antioxidant enzyme activities is a general adaptation strategy that plants use to overcome oxidative stresses (Foyer and Noctor 2003).

The enzymatic antioxidant defense system in the plant cell includes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ascorbate peroxidase (APX), glutathione reductase (GR), etc. Removing the highly toxic H₂O₂ produced during dismutation, is essential for the cell to avoid inhibition of enzymes such as those controlling the Calvin cycle in the chloroplast (Creissen et al. 1994), while the H₂O₂ produced can be scavenged by CAT and a variety of POD. CAT, which is only present in peroxisomes, dismutates H₂O₂ into the water and molecular oxygen, whereas POD decomposes H₂O₂ by oxidation of co-substrates such as phenolic compounds and/or antioxidants. Different activities of ROS scavenging enzymes including CAT, POD and PPO under stress conditions and at different growth stages have additionally been reported by Khalilzadeh et al. (2017), Das and Roychoudhury (2014) and Seysd Sharifi et al. (2017). Usually, the amount of the produced dry matter has a direct relationship with the photosynthesis efficiency of the plant and also how CO₂ fixation occurs in crops.

One of the important strategies for increasing carbon dioxide concentration in plants is using chemicals such as methanol that can increase the concentration of CO₂ in a plant and improves photosynthesis rate and growth under water deficit conditions (Ramadant and Omran 2005). Methanol is very simple alcohol, plays a significant role as a precursor in chemical synthesis and/or as a solvent. Additionally, methanol, as the less toxic compound, is used in pharmacology for drug dissolution and the food industry (Pohanka 2016). Several reports suggested that foliar applications of methanol increases CO₂ assimilation in plant’s photosynthetic pigments and helps to stabilize photosynthesis under drought stress (Ramirez et al. 2006; Ganjeali 2012). Ivanova et al. (2001) reported that the foliar application of methanol indirectly stimulates the methyltrophic bacteria that live on most plant leaves. These bacteria consume some of the methanol on the leaves and induce plant growth via auxin and cytokine production. However, the mechanism by which methanol may affect growth and yield is unknown. It is known that methanol increases stomatal conductance and decreases leaf temperature and transpiration (Makhdum et al. 2002), increases glucose metabolism and swelling pressure (Rajala et al. 1998), stem length and dry weight (Hernandez et al. 2000) and as a result, increases the plant yield. Abundant dioxide carbon supply from methanol causes the photo respiration to be shifted from catabolism to anabolism (Zebic et al. 1997). Photorespiration can be minimized with methanol spray, since 25% of carbon wastes during photorespiration (Desclaux et al. 2000). That is because methanol is absorbed in the plant and is rapidly metabolized to CO2 in the plant tissue due to the smaller size of methanol rather than CO₂ (Gout et al. 2000).

A better understanding of the antioxidant status and physiological responses of pot marigold may help the programs in which the objective is to improve the yield under drought stress. Therefore, this study aimed to evaluate the effects of methanol on physiological responses (i.e., antioxidant enzyme activity, proline, soluble carbohydrate, chlorophyll-a, chlorophyll-b, total chlorophyll, relative water content (RWC), carotenoid, stomatal conductance) of marigold under water stress conditions.

Material and Methods

A factorial experiment based on randomized complete block design with three replications was conducted under greenhouse conditions at Mohaghegh Ardabili University, Iran, in 2017. The area is located at 38° 15ʹ N latitude and 48° 15ʹ E longitude with an elevation of 1350 m above mean sea level. Experimental factors included foliar application of methanol at four levels [0 (control) (M₁), 20% (M₂), 30% (M₃) and 40% (M₄)] and four irrigation treatments [irrigation at 40 (I₁), 60 (I₂), 80 (I₃) and 100% (I₄) field capacity]. Pots were filled with a medium that contained one part sand, two parts soil and one part manure. Seeds of marigolds were prepared from Pakan Bazr Isfahan Co. and sown on 21st April in 2017. For each treatment five plants were kept in each pot. Cultural practices such as pest control, irrigation, hoeing and weeding were similar for all treatments. Foliar application of methanol was done at the beginning of flowering (60 days after sowing) in the middle of the day. The plants were watered at the field capacity until the emergence of the second leaf. At this developmental stage, water was withheld to induce water-deficit treatments. The soil humidity was measured when plants had four fully developed leaves (harvest time; the end of the experiment) by weighing soil samples and reweighing them after drying at 105 ◦C for 24 h. Soil humidity was determined at 0-20, 20-40 and beyond 40 cm depth. The measurements were done on five samples for each depth in each water treatment (Sahnoune et al. 2004). The soil was silty loam, with the pH of about 6.9. The air temperature of greenhouse ranged from 22-27 °C during the day and 18-21°C during the night. Humidity ranged from 60 to 65%.  

The leaves were detached for measuring the following characteristics at the flowering stage. The 1st to 4th youngest leaves were selected as the tissue samples. The quantum yield and stomatal conductance were measured on the uppermost fully expanded leaf using a fluorometer (chlorophyll fluorometer; Optic Science-OS-30, USA) and leaf porometer (Model SC-J Eijkelkamp, Netherlands), respectively (Kheirizadeh Arough et al. 2016). RWC was estimated gravimetrically according to the method of Tambussi et al. (2005). Chlorophyll and carotenoids were obtained based on Arnon (1949). Soluble sugars were determined based on the phenol sulphuric acid method (Dubois et al. 1956). Leaf proline content was measured according to Bates et al. (1973).

To measure the enzyme activity, 0.2 g of fresh tissue was used. To extract the protein, 0.2 g of plant’s fresh tissue was crushed by using liquid nitrogen and then 1 ml of buffer Tris-HCl (0.05 M, pH = 7.5) was added. The mixture was centrifuged for 20 min at 16000 ×g (13000 rpm) and 4 ^ºC, then the supernatant was used for the enzyme activity measurements. CAT, POD and polyphenol oxidase (PPO) activity was assayed according to Karo and Mishra (1976). To measure the above-ground biomass per plant, five plants of each pot were harvested.

Analysis of variance and comparison of means was performed using SAS computer software. The main effects of factors and their combinations were tested using the least significant difference (LSD) test.

Results

Table 1 showed a significant interaction of water limitation by methanol for the total chlorophyll, chlorophyll-a, carotenoid, Fv/Fm, leaf proline, total soluble carbohydrate, stomatal conductance, biomass, CAT, POD and PPO (Table 1). Chlorophyll-b and leaf RWC were affected by both main factors of water deficit and methanol (Table 1).

Photosynthetic pigment content

The highest chlorophyll-a and total chlorophyll content (8.16 and 13.31 mg g^-1 FW, respectively) were obtained by the 40% methanol treatment under well-watered conditions (I₁M₄). Whereas, the lowest values (3.12 and 3.99 mg g^-1 FW respectively) were observed in I₄M₁ (Table 2). Under well-watered conditions (I₁), the chlorophyll-b content was significantly higher than moderate (I₃) and severe drought stress (I₄). Reduction in chlorophyll-b in response to moderate and severe drought stress were 29.32 and 71.58%, respectively. Whereas, plants treated with 40% methanol had significantly higher chlorophyll-b values than the 20% methanol treatment.

Methanol caused increases of total chlorophyll content, from 11.50 mg g^-1 FW at no ethanol to 13.31 mg g^-1 FW at 40% methanol under well-watered conditions (Table 2). Increasing methanol concentration tor 30 and 40% decreased chlorophyll-

a, total chlorophyll and carotenoid in the moderate (I₃) and severe (I₄) drought stress conditions as compared with I₁M₀ (Table 2).

Maximum efficiency of PSII(Fv/Fm ratio)

Our findings indicated that I₁M₄ had the higher maximum quantum efficiency of PSII (0.79), whereas I₄M₁ had the lowest maximum quantum efficiency of PSII (0.32) (Table 2). Effect of severe drought stress (I₄) on Fv/Fm ratio was higher than the moderate and low drought stresses (I₃ and I₂) (Table 2).

Proline and soluble sugars

The highest (17.68 mg g^-1 F_W) and the lowest (4.12 mg g^-1 F_W) proline content belonged to I₄M₄ and I₁M₁, respectively. Proline content in plants subjected to the moderate drought stress was 22.65 and 30.76% higher than the control when 30% and 40% methanol was applied, respectively (Table 2). Severe drought stress increased the proline content by 32.26 and 44.79% as compared to the control at 30% and 40% methanol, respectively (Table 2). It seems that methanol may improve photosynthesis through an increase in the chlorophyll content (Table 2) which in turn leads to an increase in the amount of assimilates (sugars) produced (Table 2). I₄M₄ andI₄M₃ had higher total soluble carbohydrates than the other treatments.

Stomatal conductance

Our results showed that I₁M₄ had the highest (54.72 mmol/m²/s) and I₄M₁ had the lowest (40.36 mmol/m²/s) stomatal conductance among all treatments. Our data also revealed that drought and methanol application treatments significantly differed in terms of stomatal conductance (Table 2). The stomatal conductance was significantly decreased with the increase in drought stress intensity, but with increasing methanol concentration to 40%, stomatal conductance was increased. Plants treated with 30% and 40% methanol increased stomatal conductance at the moderate drought stress as much as 7.43 and 9.41%, respectively. Increased stomatal conductance due to severe drought stress at the above-mentioned methanol concentrations were 2.48 and 4.04%, respectively.

Leaf RWC

Variation of RWC values was observed in response to the drought stress intensity. The adverse effect of water stress on RWC at the severe drought stress was significantly greater than the moderate drought stress (Table 1). RWC decreased about 9.16 and 26.66% in response to moderate and severe drought stresses, respectively as compared to the control (Table 1). The highest RWC (70.40%) belonged to the foliar application of 40% methanol (Table 1) increased it by 6.84% compared to the zero ethanol treatment.

The activity of CAT, POD and PPO enzymes

The highest increase in the CAT activity (0.065 OD µg protein min^-1) was obtained at the severe drought stress plus 40% methanol (I₄M₄)_. The 10% methanol under well water irrigation (I₁M₁) had the lowest CAT activity (0.0039 OD µg protein min^-1). On the other hand, there was an increase of about 150% and 80.09% in the activity of CAT and POD enzymes, respectively, at the severe drought stress plus 40% methanol (I₄M₄) in comparison with I₄M₁ (Table 2). Increasing the intensity of drought stress led to the increased POD and PPO activity (Table 2). Maximal activities of POD (0.038 OD µg protein min^-1) and PPO (0.045 OD µg protein min^-1) activity in pot marigold plants were observed under severe drought stress plus 40% methanol (I₄M₄), while the minimum value was observed at I₁M₁ (Table 2).

Biomass

In the methanol-treated plants, the highest biomass (2.16 g plant^-1) was obtained under well water irrigation plus 40% methanol (I₁M₄), whereas the lowest biomass was obtained at the severe drought stress when no methanol a 12.9% increase in biomass under severe water stress (Table 2).

Discussion

Reduction in chlorophyll concentration is identified as a drought response mechanism to minimize the light absorption by chloroplasts (Pastenes et al. 2005). The decreased level of chlorophyll content is caused by photoinhibition and photodestruction of pigments and pigment–protein complexes and destabilization of photosynthetic membrane both induced by drought (Huseynova 2012). Methanol increases stomatal conductance, proline and soluble carbohydrate, cell swelling and chlorophyll and carotenoid content (Zbiec et al. 2003; Ramberg et al. 2002) which was corresponded with our results. Also, the foliar application of methanol increased photosynthetic capacity and dry matter. Methanol is smaller than the CO₂ and can be easily used by C₃ plants to increase dry matter and as a carbon source (Ramirez et al. 2006). Ahmadpour and Hosseinzadeh (2016) reported that increasing the methanol from 25% to 35% decreased chlorophyll content, probably due to the toxic effects of methanol at high concentrations. Since carotenoids play an important role in photoprotection (Munne-Bosch and Penuelas 2003), their increased content under the control (M₁) and the lowest concentration of methanol (M₂), indicates a higher need for photoprotection.

The Fv/Fm ratio is a suitable index for evaluating photosynthetic apparatus in plants exposed to environmental stress (Giorio 2011). Decreasing the Fv/Fm ratio is a reason for the significant effect of environmental stresses on photosynthetic efficiency caused by a decline in the transfer of electrons from PSII to PSI and light protection (Sikder et al. 2015). These findings indicate the disorganization of PSII reaction centers under water-stress conditions (Huseynova 2012). Our results showed that the application of the methanol reduced the negative effects of water shortage stress in the pot marigold plants. It is interesting to note that plant growth occurred in plants that were treated exclusively with methanol. In I₁ and I₂ irrigation levels, increasing methanol concentration led to a gradual increase of PSII efficiency. But at the moderate and severe drought stress (I₃ and I₄), increasing of methanol concentration after 30%, Fv/Fm ratio decreased. Because higher levels of methanol damage chlorophyll and PSII reaction centers, this can happen in drought, heat and light stress conditions (Yazdi Far et al. 2015). Therefore, in the present drought conditions, the improvement of photosynthesis of wheat plants under methanol application as compared with the non-methanol treatment was associated with non-stomatal factors. The results of foliar applications of methanol solution under controlled and low-stressed conditions in this study confirm prior observations reporting an increase of growth and yield in plants (Nonomura and Benson 1992). Nonimura and Benson (1992) showed that treated plants with methanol can increase net photosynthesis and improve their performance.

Our results revealed that the accumulation of proline was higher under drought and methanol treatments (Table 2). The extent of these changes was related to the intensity of the stress and concentration of methanol. This phenomenon may be part of a mechanism that prevents loss of water in the plant through osmotic adjustment. The increment of this solute coincided with low rates of maximum efficiency of PSII response to drought conditions (Table 2). In agreement with our results, others have shown that chlorophyll-a and chlorophyll -b positively correlated with RWC and stomatal conductance, but negatively correlated with carotenoid and proline contents (Ghobadi et al. 2013). Also, the lower concentration of proline during water deficit was associated with a decrease of stomatal conductance (Pompelli et al. 2010). Increased proline accumulation was reported in wheat under drought and salinity stress (Khalilzadeh et al. 2017; Seyed Sharifi et al. 2017). Proline accumulation under stressed conditions supplies energy for growth and survival and thereby helps the plant to tolerate the stress. The reduced proline oxidase may be the reason for increasing proline accumulation (Manivannan et al. 2008). However, in most cases, the osmotic adjustment was not the main consequence of proline accumulation, which was involved in other mechanisms related to sugar content and protection against oxidative damage (de Campos et al. 2011). Plants facilitate the decrease of osmotic potential and a further increase of water absorption through an increase in the soluble sugars content. Accumulation of sugars in different parts of the plants has been reported in response to environmental stresses (Prado et al. 2000; Khalilzadeh et al. 2017).

Foliar applications of methanol may also be used as an appropriate way to enhance the assimilation of CO₂ (Ganjeali, 2012). The positive impact of methanol, however, may be due to its role in reducing photorespiration and enhancing the net photosynthesis process (Nadali 2009). Since the accumulation of carbohydrates has been reported during various abiotic stresses, a decrease in sugar content as a result of the elimination of stress seems reasonable (Archbold 1940).

Foliar applications of methanol under water deficit conditions therefore may alleviate the damages caused by drought stress and also reduce the rate of stomatal conductance in plants. Our results agree with the data obtained by Khalilzadeh et al. (2017) and Boureima et al. (2012) about the decline in the stomatal conductance in plants subjected to drought stress. Makhdum et al. (2002) also reported that foliar application of methanol increased leaf thickness in cotton and this, in turn, led the plants to better maintain RWC in their leaves.

The mechanism of drought tolerance in general, and the mechanism of antioxidant production in particular, differ among species and even among cultivars of a single species. Furthermore, the form and functions of organs and tissue undergo substantial time course changes, so the capability of plants to respond to drought stress depends predominantly on the genes that are expressed at the stage of development during which the stress is imposed (Ashraf 2009).

CAT activity at all methanol concentrations was higher than the non-treated plants. The increase of CAT activity in plants under water stress was also reported in other studies (Quartacci et al. 1995; Khalilzadeh et al. 2017). Any increase in the activity of antioxidant enzymes may also be associated with the induction of antioxidant reactions which protect plants against oxidative damages. Increasing methanol concentrations increased POD activity under well-water and drought stress conditions. An increase in POD activity was also observed by different authors under drought and saline conditions (Khalilzadeh et al. 2012; Kheirizadeh et al. 2016; Babaei et al. 2017). Increased antioxidant enzyme activities due to the foliar applications of methanol may somehow indicate the alleviation of oxidative stress and the scavenging of ROS by antioxidant enzymes. Preventing the oxidative damages brought to the plant cells during drought stress has been proposed as one of the stress tolerance mechanisms and the extent of this prevention is associated with the increased antioxidant activity (Kalantar Ahmadi et al. 2015). Saruhan et al. (2012) stated that external application of the growth regulator increased antioxidant enzyme activity in the drought-tolerant maize genotypes compared to the susceptible entries.

Increasing foliar application rates of methanol under normal and stress conditions in the current study increased the pod and PPO activity under drought stress conditions. Stress tolerance in plants may be associated with their ability to scavenge ROS (Saruhan et al. 2012). According to the results obtained in the current experiment, however, the effect of methanol in alleviating the negative impacts caused by drought stress was mainly due to an increase in several enzyme activities.

Conclusions

The results showed that water stress reduced the biomass of the pot marigold plants. Methanol foliar application improved total chlorophyll, proline, soluble sugars, stomatal conductance, CAT, POD and PPO enzyme activity under normal and stress conditions. The application of 40% methanol was more effective than the other concentrations. Increasing the methanol from 30% to 40% under drought stress conditions decreased chlorophyll a,