Penconazole induced changes in photosynthesis, ion acquisition and protein profile of Mentha pulegium L. under drought stress

Abstract

Effect of penconazole (PEN) treatment on drought-stressed Mentha pulegium L. plants was investigated. Six weeks after sowing, seedlings were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity (FC) with or without PEN (15 mg l⁻¹) for 4 weeks. Results showed that the seedlings at 75 % FC showed maximum growth and water supply lower than 75 % FC was the threshold of drought-initiated negative effects on seedling growth. Drought stress significantly induced proline and carbohydrate contents and the decreased chlorophyll, photosynthesis parameters, soluble proteins and ion accumulations. Exogenous PEN increased the growth parameters, pigments, photosynthesis and ion accumulations in drought stressed and unstressed plants, but the effects of PEN were more significant under water deficit conditions. PEN also reduced the negative effects of drought by osmotic balance and protein accumulations. Electrophoretic patterns indicated that PEN treatment increased the intensity of some protein bands with the molecular weights of 30 kDa in shoot and 31 kDa in roots, and several new protein bands with the molecular masses between 116 and 14 kDa appeared in leaves, shoots and roots. These results suggest that the PEN application can be a useful tool in alleviation of effects of drought stress in M. pulegium plants.

Introduction

Drought stress is one of the most important abiotic factors limiting plant growth in arid and semiarid regions such as Iran. Mentha pulegium L. (Pennyroyal) is an aromatic and medicinal plants belonging to the Lamiaceae family present in the humid regions of Iran. It is widely used in traditional medicine, food processing, perfumery, pharmaceutical products because of the high quality of its essential oil (Shirazi et al. 2004). Like most of the cultivated plants, growth and yield of this plant decreases under water stress.

Mechanisms of drought tolerance are not yet completely clear but can be explained to some extent by stress adaptation effectors such as decrease in growth parameters, stomatal closure and decrease of photosynthesis, changes in regulatory mechanism for ion transport, changes in the accumulation and synthesis of proteins and compatible solutes (Sajedi et al. 2012; Delfine et al. 2005; Hojati et al. 2011; Safarnejad 2008). Hu et al. (2010) reported that the accumulation of 40, 31, 19 and 14 kDa dehydrin polypeptides increased with progressive water deficit, when relative water loss rate increased from 10 % to 65 % in leaves of bermudagrass. Jiang and Huang (2002) reported that the accumulation of dehydrin proteins (23, 27, 40, 42, 48, 53, and 60 kDa) was induced by progressive water deficit and ABA-treatment in fescue (F. arundinacea L.). Dehydrins are a family of hydrophilic proteins with a wide range of molecular masses (9 to 200 kDa) and they have been reported to accumulate in many plant species in response to environmental conditions (Close 1996). These proteins may protect other macromolecules or cellular structures and help in maintaining the integrity of cell membranes and proteins (Beck et al. 2007). No study has been done so far on the impact of drought stress on protein quality of M. pulegium.

Manipulation of crop production with chemicals is one of the most important advancement achieved in agriculture. The medicinal plant yield under stress can be increased by application plant growth regulators (PGRs). Penconazole (PEN) [1-(2,4-dichloro-β-propylphenethyl)-1H-1,2,4-triazole] is a triazolic group of fungicide which has plant growth regulator properties (Fletcher et al. 2000). Triazole compounds induce a variety of morphological and biochemical responses in plants including stimulation of root growth, increase of pigments, reduction in free-radical damage and increase in antioxidant potential (Kishorekumar et al. 2007; Aly and Latif 2011). These qualities make them ideal for use in increase of resistance to drought in this medicinal plant. Our previous work showed that PEN minimizes the negative effects of drought stress with evidence of less membrane damage and could be used for partial amelioration of drought stress in M. pulegium plants (Hassanpour et al. 2012). It seems important to test the effect of PEN on other biochemical parameters in order to gain more information on drought tolerance of M. pulegium. To the best of our knowledge, there is very little information available so far about the effect of triazoles on photosynthesis, ions accumulation and protein contents in medicinal plants and there was no information in M. pulegium. Therefore, the aim of the present study was to investigate the effect of drought stress on some physiological and biochemical parameters and to assess the possibility of improving drought tolerance of M. pulegium by PEN.

Materials and methods

Plant cultivation and triazole treatment

Seeds of M. pulegium L. were collected in summer 2009 from Chalous (Province of Mazandaran, Iran). Seeds were sown in Tref peat, in a growth chamber with 16 h light/8 h dark period and day/night temperatures of 25/18 °C. Then, the seedlings were thinned to five per pot, 6 weeks after sowing. The pots were filled with sandy-loam soil (the % FC of soil was 11.8 (w/w) and pH 6.8). The pots were divided into eight groups of six pots each and then were irrigated to 100, 75, 50 and 25 % FC with or without 15 mg l⁻¹ PEN and the water supplies were adjusted by weighting the pots. The PEN was applied uniformly to the plants as a fine spray using an atomizer once a week for 4 weeks. Four weeks after water deficit, four plants per treatment (four replicates) were collected for analyses in all the experiments.

Plant growth and pigment contents

The plants were evaluated after 4 weeks of drought stress in terms of fresh weight and dry weight. The RGR and NAR calculated from the following formula, where W₁ and W₂ are mean weights, and LA₁ and LA₂ of harvest are mean leaf areas at harvest times t₁ and t₂, respectively (Evans and Hughes 1962).

$$\mathrm{RGR}={log}_{\mathrm{e}}{\mathrm{W}}_2-{log}_{\mathrm{e}}{\mathrm{W}}_1/{\mathrm{t}}_2-{\mathrm{t}}_1$$

$$\mathrm{NAR}=\left({log}_{\mathrm{e}}{\mathrm{LA}}_2-{log}_{\mathrm{e}}{\mathrm{LA}}_1/{\mathrm{t}}_2-{\mathrm{t}}_1\right)\times \left({\mathrm{W}}_2-{\mathrm{W}}_1/{\mathrm{LA}}_2-{\mathrm{LA}}_1\right)$$

Chlorophyll was estimated by the method of Arnon (1949), carotenoid content was calculated using the formula of Kirk and Allen (1965) and expressed in mg g⁻¹ fresh weight.

Photosynthetic parameters

The net photosynthetic rate (P_n), internal CO₂ concentration (C_i), stomatal conductance (gs) and transpiration rate (E) were measured from fully expanded leaves using a portable infrared gas analyzer (IRGA, LCA4, ADC Bio. Scientific Ltd., Herfordshire, UK): leaf surface area 1 cm², ambient CO₂ concentration 370 μmol mol⁻¹ and PPFD 200 μmol m⁻² s⁻¹.

Ions content

Samples were finely ground, and 0.5 g plant material was digested with 5 ml sulphuric acid in a digestal system (23-130-20-21 Model). The concentrations of Ca²⁺ and K⁺ were analyzed by ICP-ODS (Vista-MPX) (Nyomora et al. 1997).

Proline and carbohydrate contents

Free proline was determined by the method of Bates et al. (1973). Approximately 0.5 g of plant material was homogenized for 5 min in 5 ml of 3 % aqueous sulphosalicylic acid. Two ml of the extract was kept with 2 ml of acid ninhydrin and 2 ml of glacial acetic acid for 1 h at 100 °C. The reaction mixture was extracted with 4 ml toluene and the absorbance was read at 520 nm. For determination of saccharides content, 0.5 g of dry powder was extracted using 10 ml of 80 % (v/v) ethanol, and supernatant was collected after centrifugation at 1480 g for 15 min two times. The residue from ethanol extraction was subsequently used for polysaccharide extraction by boiling water. Total saccharides content was estimated by the method of Dubois et al. (1956). Reducing saccharides were quantified according to Nelson (1944). Oligosaccharide content was obtained from difference between soluble and reducing saccharides contents.

Protein extraction and quantification

For estimation of total protein content, 0.5 g fresh sample was homogenized at 4 °C with a mortar in 1 M Tris–HCl (pH 6.8). The homogenates were centrifuged at 13000 g for 30 min two times at 4 °C using a Heraeus 400R microfuge. Supernatant was kept at −70 °C and used for protein determination and enzyme assay. Protein concentration was measured according to Bradford (1976), using bovine serum albumin (BSA) as standard. Five milliliters of the Bradford reagent and 100 μl of the each protein extract were mixed and then reaction mixtures were incubated at room temperature for 20 min. The absorbance values were measured at 595 nm using UV-visible spectrophotometer (UV-160, Shimadzu, Tokyo, Japan). For determination of protein patterns, SDS-PAGE was performed according to Laemmli (1970) using 12 % acrylamide. For detection of proteins, gels were stained with 0.03 % Comassie Briliant Blue G250.

Statistical analysis

Analysis of variance was conducted with one − way ANOVA test using SPSS 18.0, and means were compared by Duncan tests at P ≤ 0.05 level. Each experiment was repeated four times.

Results

Effect of PEN on growth parameters under drought stress

The dry weight, RGR and NAR parameters increased slightly at 75 % FC and then decreased remarkably at lower levels of FC (Fig. 1). In preliminary experiments, the seedlings were treated with different concentrations (0, 5, 10, 15 and 20 mg l⁻¹) of PEN to determine the optimum concentration desired. Among these concentrations 15 mg l⁻¹ of PEN significantly increased the dry weight (unpublished data). Hence, 15 mg l⁻¹ PEN was used for this study. PEN treatment significantly increased the plant dry weight, NAR and RGR in both drought-stressed and unstressed plants (Fig. 1).

Fig. 1.

Effect of PEN on fresh weight (a), dry weight (b), NAR (c) and RGR (d) of M. pulegium under different irrigation levels (100, 75, 50 and 25 % FC) after 4 weeks. Vertical bars indicate mean ± SE of four replicates. Different letters above columns indicated significant (P ≤ 0.05) differences. PEN Penconazole, FC Field capacity, RGR Relative growth rate, NAR Net assimilation rate

Effect of PEN on pigment contents under drought stress

Some physiological and biochemical changes in M. pulegium were monitored under drought stress and PEN treatment. Pigment content analysis indicated that drought stress significantly decreased the levels of chlorophyll a (Chl a), chlorophyll b (Chl b) and also increased carotenoid (Car) and Car/Chl ratio in leaves (Table 1). PEN treatment to drought-stressed and unstressed plants increased Chl a, Chl b and Car contents in leaves when compared to without PEN, while effect of PEN were more significant under drought stress as compared to unstressed plants. At 25 % FC with PEN, the contents of Chl a, Chl b and Car were 228, 126 and 140 % higher than that of drought stress without PEN, respectively (Table 1). Car/Chl ratio significantly decreased 20.2 and 14.9 % at 50 and 25 % FC with PEN as compared to without PEN, respectively (Table 1). These results indicate that Chl a, which is the main pigment of photosynthesis increased more than Chl b and Car under drought stress with PEN.

Table 1.

Effects of PEN on pigment contents of M. pulegium under different irrigation levels

Parameters	PEN (mg l−1)	Field capacity (%)
		100	75	50	25
Chl a (mg g−1 FW)	0	0.31 ± 0.025 e	0.36 ± 0.041 d	0.23 ± 0.039 f	0.12 ± 0.046 g
	15	0.34 ± 0.031 e	0.42 ± 0.026 b	0.54 ± 0.053 a	0.38 ± 0.032 c
Chl b (mg g−1 FW)	0	0.23 ± 0.016 bc	0.22 ± 0.021 c	0.11 ± 0.025 e	0.09 ± 0.025 ef
	15	0.26 ± 0.011 b	0.34 ± 0.016 a	0.27 ± 0.031 b	0.21 ± 0.020 c
Car (mg g−1 FW)	0	0.08 ± 0.023 f	0.06 ± 0.016 f	0.12 ± 0.029 e	0.15 ± 0.017 cd
	15	0.13 ± 0.018 de	0.14 ± 0.025 cd	0.23 ± 0.031 b	0.36 ± 0.021 a
Car/Chl	0	0.132 ± 0.034 f	0.106 ± 0.026 f	0.352 ± 0.050 c	0.710 ± 0.033 a
	15	0.216 ± 0.045 e	0.181 ± 0.040 e	0.281 ± 0.035 d	0.604 ± 0.024 b

PEN Penconazole, Chl Chlorophyll, Car Carotenoid

Values are means ± SE of four replicates. Different letters indicated significant (p ≤ 0.05) differences

Effect of PEN on photosynthetic parameters under drought stress

Drought stress significantly decreased net photosynthetic rate (P_n), transpiration rate (E), stomatal conductance (g_s) and intercellular CO₂ (C_i) in M. pulegium plants (Table 2). PEN treatment to drought stressed plants increased P_n and C_i at all the % FC levels significantly and decreased E and g_s as compared to without PEN. At 50 % FC with PEN, P_n and C_i were 31.6 and 13.98 % higher than that of without PEN, and E and g_s were 18.9 and 12.5 % lower than that of without PEN, respectively. Effect of PEN to unstressed plants slightly increased P_n and C_i and decreased E and g_s as compared to stressed plants (Table 2).

Table 2.

Effects of PEN on photosynthetic parameters of M. pulegium under different irrigation levels

Parameters	PEN (mg l−1)	Field capacity (%)
		100	75	50	25
Pn (μmol CO2 m−2 s−1)	0	8.32 ± 0.29 d	8.52 ± 0.46 cd	6.93 ± 0.29 e	2.24 ± 0.49 f
	15	9.91 ± 0.71 b	11.26 ± 0.61 a	9.12 ± 53 bc	3.25 ± 0.31 f
E (mmol H2O m−2 s−1)	0	4.01 ± 0.33 a	4.18 ± 0.36 a	3.49 ± 0.42 abc	3.69 ± 0.12 abc
	15	3.94 ± 0.14 ab	3.22 ± 0.21 cd	2.83 ± 0.36 d	3.28 ± 0.29 bcd
gs (mol H2O m−2 s−1)	0	0.81 ± 0.018 b	0.85 ± 0.042 a	0.56 ± 0.033 d	0.44 ± 0.02 e
	15	0.78 ± 0.036 bc	0.76 ± 0.017 c	0.49 ± 0.032 e	0.32 ± 0.025 f
Ci (mgl−1)	0	238.9 ± 8.07 b	244.4 ± 6.35 b	216.6 ± 5.73 c	97.9 ± 3.43 e
	15	249.3 ± 5.27 b	268.2 ± 5.02 a	246.9 ± 5.33 b	127.6 ± 6.91 d

PEN Penconazole, FC Field capacity, P _n Net photosynthetic rate, E Transpiration rate, gs Stomatal conductance, C _i Internal CO₂ concentration

Values are means ± SE of four replicates. Different letters indicated significant (p ≤ 0.05) differences

Effect of PEN on Ca²⁺ and K⁺ ions content under drought stress

M. pulegium plants cultivated at different %FC levels showed higher amount of K⁺ and Ca²⁺ in roots as compared to leaves (Fig. 2). Ions contents significantly decreased under drought stress and increased by PEN when compared to control plants. PEN treatment to drought-stressed plants increased the K⁺ content in leaves and roots as compared to without PEN (ca. 102.8 % and 10 % increase at 25 % FC in the leaves and roots, respectively). In unstressed plants, K⁺ contents slightly decreased by PEN as compared to control (Fig. 2a). PEN treatment significantly increased Ca²⁺ contents in drought stressed M. pulegium plants as compared to without PEN (ca. 49.8 % and 89.2 % increase at 25 % FC in the leaves and roots, respectively). In unstressed plants, Ca²⁺ contents slightly decreased after treatment with PEN as compared to control (Fig. 2b).

Fig. 2.

Effect of PEN on K⁺ (a) and Ca²⁺ (b) contents of M. pulegium under different irrigation levels (100, 75, 50 and 25 % FC) after 4 weeks. Vertical bars indicate mean ± SE of four replicates. Different letters above columns indicated significant (P ≤ 0.05) differences. PEN Penconazole, FC Field capacity

Effect of PEN on proline and carbohydrate contents under drought stress

Drought stress significantly increased proline content in the leaves and roots of M. pulegium at 50 and 25 % FC (Fig. 3). PEN treatment to drought stressed caused induction of this parameter to a significant level at 75, 50 and 25 % FC in leaves and 50 and 25 % FC in roots. The highest amount of free proline content in leaves was detected at 25 % FC under PEN treatment, which was 37.7 % higher than that of without PEN treatment (Fig. 3). Drought stress caused a significant rise in the contents of reducing saccharides (RS), oligosaccharides (OS), soluble saccharides (SS) and a significant decline in polysaccharide (PS) content in leaves and roots of M. pulegium plants (Table 3). The carbohydrate contents in leaves were higher than that of roots. PEN treatment to drought-stressed plants caused more induction in sugar contents as compared to without PEN (ca. 135.7 %, 17.6 %, 37.3 % and 41.2 % increase at 25 % FC for RS, OS, SS and PS in leaves, respectively).

Fig. 3.

Effect of PEN on the proline content of M. pulegium under different irrigation levels (100, 75, 50 and 25 % FC) after 4 weeks. Vertical bars indicate mean ± SE of four replicates. Different letters above columns indicated significant (P ≤ 0.05) differences. PEN Penconazole, FC Field capacity

Table 3.

Effects of PEN on carbohydrate contents of M. pulegium under different irrigation levels

Parameters	Organs	PEN (mg l−1)	Field capacity (%)
			100	75	50	25
RS (%DW)	L	0	0.22 ± 0.072 f	0.36 ± 0.106 e	0.53 ± 0.091 e	0.84 ± 0.120 d
		15	0.49 ± 0.122 e	0.94 ± 0.195 c	1.58 ± 0.139 b	1.98 ± 0.108 a
	R	0	0.11 ± 0.024 e	0.27 ± 0.035 d	0.33 ± 0.059 d	0.55 ± 0.099 c
		15	0.25 ± 0.059 d	0.64 ± 0.069 c	0.80 ± 0.042 b	1.15 ± 0.079 a
OS (%DW)	L	0	1.38 ± 0.198 f	1.93 ± 0.416 de	2.55 ± 0.195 d	4.19 ± 0.180 b
		15	1.67 ± 0.313 ef	2.22 ± 0.284 de	3.37 ± 0.337 c	4.93 ± 0.227 a
	R	0	0.84 ± 0.346 g	1.43 ± 0.413 ef	1.95 ± 0.241 cd	2.09 ± 0.358 b
		15	1.06 ± 0.218 fg	1.63 ± 0.296 de	2.48 ± 0.391 bc	2.87 ± 0.262 a
SS (%DW)	L	0	1.60 ± 0.237 e	2.29 ± 0.327 d	3.08 ± 0.487 c	5.03 ± 0.132 b
		15	2.16 ± 0.575 d	3.16 ± 0.407 c	4.95 ± 0.536 b	6.91 ± 0.257 a
	R	0	0.95 ± 0.589 g	1.70 ± 0.494 e	2.29 ± 0.301 cd	2.64 ± 0.417 c
		15	1.36 ± 0.327 f	2.27 ± 0.325 d	3.28 ± 0.299 b	4.02 ± 0.227 a
PS (%DW)	L	0	2.53 ± 0.216 a	2.04 ± 0.199 b	1.32 ± 0.321 c	0.97 ± 0.095 d
		15	2.64 ± 0.149 a	2.46 ± 0.232 a	1.90 ± 0.232 b	1.37 ± 0.183 c
	R	0	1.23 ± 0.289 b	0.56 ± 0.274 d	0.32 ± 0.231 ef	0.23 ± 0.069 f
		15	1.42 ± 0.218 ab	1.49 ± 0.204 a	0.93 ± 0.364 c	0.52 ± 0.146 de

L Leaf, R Root, PEN Penconazole, FC Field capacity, RS Reducing saccharides, OS Oligosaccharides, SS Soluble saccharides, PS Polysaccharides, TS Total saccharides

Values are means ± SE of four replicates. Different letters indicated significant (p ≤ 0.05) differences

Effect of PEN on proteins content and profile under drought stress

The protein contents significantly changed under drought stress and PEN treatment. At 75 % FC, the total soluble protein content was maximum in all the organs and then decreased gradually at 50 and 25 % FC (Fig. 4). PEN treatment to drought stressed and unstressed plants increased this parameter in all the organs, while effects of PEN were more significant in drought stressed as compared to unstressed plants in M. pulegium. According to SDS-PAGE analysis of soluble protein patterns in leaves, several protein bands were observed under drought stress and the intensity of bands decreased at the lower level of %FC (Fig. 5a). The strongest protein band with the molecular mass about 37 kDa was observed in all treatments. PEN treatment to drought-stressed and unstressed plants increased intensity and number of protein bands. Three new bands with the molecular masses of 21, 17 and 14 kDa appeared by PEN treatment at 75 and 50 % FC, and then disappeared at 25 % FC (Fig. 5a). In shoot, intensity of a protein band with the molecular mass of 30 kDa significantly increased and some new bands with the molecular masses of 116, 44, 37, 21 and 17 kDa appeared at 25 % FC (Fig. 5b). PEN treatment increased the intensity of a 30 kDa protein band and showed some new bands with the molecular masses of 116, 58.2, 44, 37, 21, 17 and 14 kDa at 75, 50 and 25 % FC in shoot (Fig. 5b). It seems that PEN caused induction of stress proteins even at higher % FC level (75 % FC). In roots, intensity of some proteins with the different molecular masses was affected by drought stress in roots (Fig. 5c). At 50 % FC, the intensity of band with the molecular mass of 31 kDa increased and two new protein bands (65 and 54 kDa) appeared weakly and then the intensity of all of them decreased at 25 % FC. PEN treatment to drought-stressed and unstressed plants increased the intensity of protein band with the molecular mass of 31 kDa and showed two new bands with the molecular mass of 65 and 54 kDa at all the %FC levels (Fig. 5c).

Fig. 4.

Effect of PEN on the protein content of M. pulegium under different irrigation levels (100, 75, 50 and 25 % FC) after 4 weeks. Vertical bars indicate mean ± SE of four replicates. Different letters above columns indicated significant (P ≤ 0.05) differences. PEN Penconazole, FC Field capacity

Fig. 5.

SDS-PAGE profiles of leaf (a), shoot (b), root (c) proteins of M. pulegium plants under 100, 75, 50 and 25 % FC with (+) or without 15 mgl⁻¹ PEN (−). Arrows indicate some of the affected bands. PEN Penconazole, FC Field capacity, M Protein marker (kDa)

Discussion

Plant adaptations to drought stress are complex and affected by internal constitutive drought tolerance mechanisms and external environmental factors. In our experiment, plant growth was significantly inhibited in M. pulegium when subjected to the drought stress (Fig. 1). There was no significant difference between 100 and 75 % water stress level, it appears that 75 % FC might be a suitable treatment for M. pulegium growth. Application of PEN ameliorated the adverse effects of water stress by increasing of growth parameters in M. pulegium (Fig. 1). Similar results were reported in Matricaria chamomilla (Hojati et al. 2011) and alfalfa (Medicago sativa L.) (Safarnejad 2008) under other triazole treatments. The increase of the dry weight, RGR and NAR by PEN could be the result of the ability of triazoles to increase the cytokinin content (Grossman 1990) and cell division.

Drought stress caused a decrease in Chl contents and an increase in Car contents when compared to control plants (Table 1). The chlorophyll reduction under drought stress has been attributed to the inhibition of chlorophyll synthesis as well as to accelerated turnover of chlorophyll already present (Reddy and Rao 1968). Application of PEN to drought stressed and unstressed plants increased the chlorophyll content in M. pulegium leaves. This result is consistent with the findings in Solenostemon rotundifolius and wheat (Triticum aestivum L.) under other triazole compounds (Kishorekumar et al. 2007; Aly and Latif 2011). It has been hypothesized the higher chlorophyll content in PEN-treated plants could be the result of triazole influence on the endogenous cytokinin content. Although the change in cytokinin concentration was not evaluated in the present work, it can be assumed that the observed induction in chlorophyll content could probably interfere with increased cytokinin content under PEN that in turn enhances chloroplast differentiation and chlorophyll biosynthesis, and prevents chlorophyll degradation (Fletcher et al. 2000). Veerasamy et al. (2007) also showed that application of the zeatin riboside helped to maintain higher chlorophyll concentration and photochemical efficiency under heat stress.

Drought stress significantly decreased photosynthetic parameters in M. pulegium plants. Ranjbarfardooei et al. (2000) reported that net photosynthetic rates decreased with increasing drought stress in Pistacia khinjuk and P. mutica. Pompelli et al. (2010) also showed that net photosynthesis and stomata conductance decreased under drought stress in Jatropha curcas. PEN treatment to drought stressed and unstressed plants increased P_n and C_i in M. pulegium plants (Table 2). Induction of photosynthesis by PEN has been attributed to increase of RUBP-carboxylase activity by triazoles (Yan and Pan 1992). Triazoles inhibit cytochrome P-450 mediated monooxygenases which inhibit the synthesis of GA and the ABA degradation to phaseic acid (Rademacher 1997). It seems that the decrease of E and the increase of C_i by PEN in M. pulegium may be related to higher accumulation of ABA, partial closure of stomata and increased intercellular CO₂ concentration in PEN treated plants.

Drought stress caused a significantly decrease in Ca²⁺ and K⁺ concentrations in leaves and roots of M. pulegium plants, while PEN increased both Ca²⁺ and K⁺ concentrations (Fig. 2). This result is consistent with previous reports in alfalfa (Medicago sativa L.) under osmotic stress (Safarnejad 2008). Our previous work in M. pulegium showed a significant increase in the electrolyte leakage under drought stress and a decline by PEN treatment (Hassanpour et al. 2012). It seems that the ability of PEN to ameliorate the negative effects of drought stress on growth may be due to decrease of electrolyte leakage and an increase of accumulation of ions in the leaves and roots of M. pulegium.

Proline content was remarkably increased under drought stress and PEN treatment in M. pulegium (Fig. 3). Proline is an osmolyte that may play a significant role in maintaining leaf osmotic potential, enabling plants to maintain turgor when water is limiting or soil salinity is high (Sadiqov et al. 2002). Proline may protect protein configurations during dehydration. It also has been observed a positive relation between proline accumulation and drought tolerance (Aly and Latif 2011). Exogenously applied proline has been shown to significantly increase the stress tolerance of plants (Santarius 1992). In this research, PEN treatment significantly increased proline content, especially in drought stressed plants. Triazole could induce a transient rise in the ABA content (Jaleel et al. 2008). Increased ABA content due to the triazole treatment could be the main reason for the increased proline content in the PEN treated M. pulegium plants. Also, Khedr et al. (2003) showed that exogenous proline increased the protein content in Pancratium maritimum L. under salt stress. With the exception of these experiments, few studies have observed the direct effect of triazoles on proline accumulation within plant tissue and a subsequent increase on protein content (Hojati et al. 2011). Since qualitative and quantitative study on proteins was evaluated in the present work, we suggest that proline accumulation could probably require for induction of new stress tolerance protein by PEN. Perhaps this might depend on the experimental tissue or its stage of development.

The increase of carbohydrate content found in drought stressed M. pulegium agrees with results reported on other plants (Table 3). Increase in sugar content has been observed in chickpea (Mafakheri et al. 2011), Dactylis glomerata and Poa bulbosa (Volaire et al. 2001) under drought stress. The high carbohydrate concentrations, beside their role in decreasing the water potential, contributed both to the prevention of oxidative damage and to the maintenance of the structure of proteins and membranes under severe dehydration. The hydroxyl groups of sugars may substitute for water to maintain hydrophilic interactions in membranes and proteins during dehydration and prevent protein denaturation (AL-Rumaih and AL-Rumaih 2007). In the present study, PEN treatment to drought stressed plants induced higher amount of carbohydrates in comparison to without PEN. The higher amount of carbohydrate under PEN treatment could be result of induction of chlorophyll accumulation and the photosynthesis rate in M. pulegium and might help to maintenance of the structure of proteins under stress.

The total protein content decreased in all the organs of M. pulegium under drought stress and drastically increased by PEN treatment (Fig. 4). Several studies were reported about the protein reduction under severe drought stress, for example in maize (Mohammadkhani and Heidari 2008), bermudagrass (Hu et al. 2010) and chickpea (Mafakheri et al. 2011). The reduction in protein content under water deficit may be due to the decrease in protein synthesis, the decrease in the availability of amino acids and the deaturation of enzymes involved in protein synthesis (Levitt 1980). In our experiment, PEN-treated M. pulegium maintained higher total soluble protein content than non- PEN treated plants. Increased protein contents by PEN may be associated with the higher production of cytokinins under triazole treatment (Fletcher et al. 2000). Drought-stressed M. pulegium showed lower chlorophyll contents, and treatment with PEN resulted in higher chlorophyll contents. It has also been reported that chlorophyll loss is linked to protein degradation (Hashimoto et al. 1989). These results suggest that PEN may alleviate the negative effect of drought by modulation of growth regulators level, increase of pigments and inhibition of protein degradation.

Severe drought stress increased the intensity of protein bands with the molecular weight of 30 kDa in shoot and 31 kDa in root of M. pulegium and PEN treatment under water deficit conditions caused to more induction of these bands (Fig. 5b, c). There was no study about the impact of drought stress on protein quality of M. pulegium. Similar size dehydrins were also observed in other plants, dehydrins of 31 kDa were detected in leaves of bermudagrass induced by drought and ABA-treatment (Hu et al. 2010). The accumulation of a 30 kDa protein has also been reported in leaves of Zea mays under drought stress (Mohammadkhani and Heidari 2008). The 30-kDa chlorophyll-a/b-binding protein as a Ca²⁺-binding protein located in photosystem II and involved in water oxidation (Britt 1996). Our results suggest that the upregulation of 30 kDa proteins in PSII by application of PEN could help to increased photochemical efficiency, as indicated by a higher photosynthesis, RGR and NAR in PEN-treated than untreated plants in M. pulegium. At 25 % FC, drought stress induced the synthesis of some new proteins with the molecular weights of 116, 44, 37, 21 and 17 kDa in shoots, and two new faint protein bands with 65 and 54 kDa in roots (Fig. 5b, c). PEN treatment to drought-stressed plants caused induction of these bands even in higher levels of %FC (at 75 and 50 % FC). This is the first report about the effect of triazoles on protein quality and this research is the first study. Volaire et al. (2001) found that dehydrin like proteins of 22, 32, 42, and 44 kDa were expressed in Dactylis plants exposed to drought stress, but not detected in well-watered plants. Mohammadkhani and Heidari (2008) showed the accumulation of dehydrin-like 37, 20 and 17 kDa proteins in leaves, and 57 and 65 kDa in roots increased in maize under drought stress. In the present study, the presence of protein bands only in severe drought suggests a probable role of these proteins in drought tolerance and this family of dehydrins may be associated with dehydration tolerance in M. pulegium. The specific functions of these dehydrins involved in drought adaptation are still unclear, despite findings of previous studies that suggest the accumulation of dehydrins in response to water deficit may help control water loss through osmotic protection of cells from further dehydration during drought stress (Volaire 2002). PEN treatment to drought-stressed and unstressed plants induced some new protein bands in all the organs, while the effect of PEN was more significant in drought stressed as compared to unstressed plants. It seems that PEN under water deficit conditions may induce some proteins, which may be involved in adaptation to drought stress. PEN treatment induced some new protein bands with the molecular weights of 21, 17 and 14 kDa in leaves at 75 and 50 % FC. Dehydrin-like proteins with the similar molecular weights were reported under drought in maize (Mohammadkhani and Heidari 2008). Appearance of new proteins at higher %FC levels (75 and 50 % FC) by PEN suggest that PEN treatment could help to develop the drought tolerance genotypes and contribute toward drought tolerance in M. pulegium plants.

In conclusion, physiological and biochemical analyses have shown that 75 % FC might be a suitable treatment for achieving efficient yield in M. pulegium. A possible survival strategy of plants under water deficit condition is induction of secondary metabolism that could alleviate the deleterious effect of drought stress. PEN treatment increased growth parameters, photosynthesis, pigments, protein, ion concentrations and maintained the osmotic balance of M. pulegium. Our results suggest that PEN can be used as a potential tool to develop plant tolerance and to alleviate drought stress in medicinal plant M. pulegium.

Abbreviations

PEN

Penconazole

FC

Field capacity

RGR

Relative growth rate

NAR

Net assimilation rate

Chl

Chlorophyll

Car

Carotenoid

SDS-PAGE

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis