Scholarly article on topic 'Physiological and biochemical mechanisms of allelochemicals in aqueous extracts of diploid and mixoploid Trigonella foenum-graecum L.'

Physiological and biochemical mechanisms of allelochemicals in aqueous extracts of diploid and mixoploid Trigonella foenum-graecum L. Academic research paper on "Biological sciences"

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South African Journal of Botany
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{Fenugreek / "Ploidy level" / "Developmental stage" / "Mode of action" / "Aqueous extract" / Lettuce}

Abstract of research paper on Biological sciences, author of scientific article — Faten Omezzine, Afef Ladhari, Rabiaa Haouala

Abstract This study was conducted to evaluate the effect of shoot aqueous extracts of diploid and mixoploid fenugreek at vegetative, flowering and fruiting stages on some physiological and biochemical processes in lettuce. The allelochemicals stress was registered as the result of aqueous extract application, which was added to the Hoagland nutrient solution at concentration corresponding to IC50 (50% inhibition of germination or root growth). The germination inhibition seems to be correlated with membrane deterioration as proved by a strong electrolyte leakage, increase in malondialdehyde (MDA) content, and mitochondrial respiration disruption due to a decrease in dehydrogenases activity. These disruptions were recorded with all test extracts, especially fruiting stage extract of diploid and mixoploid plants. For seedling growth inhibition, the roots showed the same interference, especially in the presence of aqueous extract of plant material harvested at the vegetative stage for diploid and at flowering of mixoploid plants. Chlorophyll content was slightly reduced while carotenoid content was significantly reduced. The lettuce seedlings have circumvented the allelochemicals stress, by i) increasing the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activity, ii) accumulation of proline and iii) the production of secondary metabolites with antioxidant potent, such as polyphenols, flavonoids and alkaloids. The importance of these phenomenons varied with the extract origin and target organ, which is in favor of speculating on the allelochemicals specificity and on the change in the chemical composition of different extracts. Also, understanding of the different mechanisms of allelochemicals may provide a basis for the development of growth regulators and natural pesticides to boost up production in sustainable agriculture.

Academic research paper on topic "Physiological and biochemical mechanisms of allelochemicals in aqueous extracts of diploid and mixoploid Trigonella foenum-graecum L."

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Physiological and biochemical mechanisms of allelochemicals in aqueous extracts of diploid and mixoploid Trigonella foenum-graecum L.

Faten Omezzine a'*, Afef Ladharia, Rabiaa Haouala b

a Department of Biology, Faculty of Sciences ofBizerte, University of Carthage, Amilcar, 1054, Tunisia, (UR13AGR05) b Department of Biological Sciences and Plant Protection, Higher Agronomic Institute of Chott-Mariem, University ofSousse, Chott-Mariem, 4042, Sousse, Tunisia, (UR13AGR05)

ARTICLE INFO ABSTRACT

This study was conducted to evaluate the effect of shoot aqueous extracts of diploid and mixoploid fenugreek at vegetative, flowering and fruiting stages on some physiological and biochemical processes in lettuce. The allelochemicals stress was registered as the result of aqueous extract application, which was added to the Hoagland nutrient solution at concentration corresponding to IC50 (50% inhibition of germination or root growth). The germination inhibition seems to be correlated with membrane deterioration as proved by a strong electrolyte leakage, increase in malondialdehyde (MDA) content, and mitochondrial respiration disruption due to a decrease in dehydrogenases activity. These disruptions were recorded with all test extracts, especially fruiting stage extract of diploid and mixoploid plants. For seedling growth inhibition, the roots showed the same interference, especially in the presence of aqueous extract of plant material harvested at the vegetative stage for diploid and at flowering of mixoploid plants. Chlorophyll content was slightly reduced while carotenoid content was significantly reduced. The lettuce seedlings have circumvented the allelochemicals stress, by i) increasing the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activity, ii) accumulation of proline and iii) the production of secondary metabolites with antioxidant potent, such as polyphenols, fla-vonoids and alkaloids. The importance of these phenomenons varied with the extract origin and target organ, which is in favor of speculating on the allelochemicals specificity and on the change in the chemical composition of different extracts. Also, understanding of the different mechanisms of allelochemicals may provide a basis for the development of growth regulators and natural pesticides to boost up production in sustainable agriculture.

© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

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Article history:

Received 10 March 2014

Received in revised form 10 April 2014

Accepted 16 April 2014

Available online xxxx

Edited by J Van Staden

Keywords: Fenugreek Ploidy level Developmental stage Mode of action Aqueous extract Lettuce

1. Introduction

Environmental stress factors limit the agricultural productivity and many of these factors are related to the metabolic processes. The plant response to stress depends on the duration, severity and rate of imposed stress (Munne-Bosch and Alegere, 2004). Under natural conditions, multiple stresses develop progressively and gradually, eliciting morphological, physiological and biochemical responses. Plants produce various secondary metabolites some of them are known to be allelochemicals, whose action can be beneficial or detrimental to the growth and development of the receptor species. In the latter case, the effect is described by a biotic stress "allelochemical stress" (Pedrol et al., 2006). Indeed, it is reported that allelochemicals reduce cell division (Sanchez-Moreiras et al., 2008) directly by affecting many physiological and biochemical reactions (Einhellig, 2002). Therefore, they influence on the growth and development of plants (Lara-Nunez et al.,

* Corresponding author at: Higher Agronomic Institute of Chott-Mariem, BP 47,4042 Chott-Mariem, Sousse, Tunisia. Tel.: +216 97080195; fax: +216 73327591. E-mail address: faten.omez@yahoo.fr (F. Omezzine).

2009). According to Einhellig (1986, 1995) and Macias et al. (2001), specifically to a given allelopathic compound mode of action has not yet been studied and a lot of additional information is required. One of the most important limitations that reduced attempts to learn about how allelochemicals affect the growth of the receiving plant is the lack of sufficient of composed quantities for to study the effects on physiological processes and cellular mechanisms (Einhellig, 1995). In addition, difficulties also limit these studies that come from the multitude of potential molecular targets (Einhellig, 1986). Thus, allelochemicals have several molecular targets and are known to effect many cellular processes in target plants, viz. stomatal closure (Barkosky et al., 2000), cell division (Anaya and Pelayo-Benavides, 1997), membrane permeability (Galindo et al., 1999), absorption of nutrients (Baar et al., 1994), photosynthesis (Baziramakenga et al., 1994), respiration (Abrahim et al., 2000), transpiration, efficiency of photosystem II (PSII), the synthesis of ATP, the phytohormone metabolism, the production of reactive oxygen species, gene expression and other metabolic processes (Blum, 2005).

Previous studies (Omezzine and Haouala, 2013; Omezzine et al., 2014) have shown that different extracts of the aerial parts of diploid and mixoploid T. foenum-graecum, were toxic for lettuce germination and growth. The phytotoxicity degree was largely dependent on the

http: //dx.doi.org/10.1016/j.sajb.2014.04.009

0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

developmental stage at which the material was collected, but also with the ploidy level. In this study, the aqueous extracts of Trigonellafoenum-graecum (diploid and mixoploid) were used as a factor of biotic stress on lettuce and our aim is to compare their effects on a number of biochemical and physiological parameters in lettuce to understand their mechanisms of germination and growth inhibition. The parameters evaluated are: the secondary metabolites production, lyase enzyme activity, cell metabolic activity, content of photosynthetic pigments and membrane integrity assessed by measuring the electrolyte leakage and the lipid peroxidation.

2. Material and methods

2.1. Plant material and mixoploidy induction

The mixoploid plants of T. foenum-graecum were obtained following seed treatment with 0.05% colchicine solution, according to Omezzine et al. (2012). Fenugreek treated and untreated seeds were sown in field under natural conditions in March 2011. The mixoploidy confirmation was done by flow cytometry and stomata and pollen grain size (Omezzine et al., 2012). Aerial parts of diploid (plant from untreated seeds) and mixoploid (plant from colchicine treated seeds) plants were harvested at vegetative (plants with 8 leaves), flowering (50% of flowers are blossomed) and fruiting stages (50% of the pods have reached a typical length). Fresh plants were washed out under tap water, then oven-dried at 60 °C for 72 h, powdered and used for extraction.

2.2. Lettuce growth and treatment conditions

Seeds were germinated in Petri dishes at room temperature in the dark. Seven-day old seedlings were irrigated with distilled water during the first week. Uniform seedlings were subsequently cultured individually in a hydroponic system containing a complete Hoagland's medium (Hoagland and Arnon, 1950) diluted eightfold in a greenhouse (16 h light/8 h dark at 20/17 °C). After two weeks, the plants were divided into batches cultured in the absence (control group) and in the presence of aqueous extracts of diploid and mixoploid fenugreek plants, harvested at the vegetative, flowering and fruiting stage, and prepared at a concentration inducing a reduction of 50% root growth (IC50). The culture media were aerated continuously and the renewal was done every 2 days. At the end of the treatment period (7 days), the plants were harvested and separated into leaves and roots.

2.3. Electrolyte leakage

The electrolyte leakage (EL) was determined as described by Lutts et al. (1996). Seeds or roots of fresh lettuce seedlings were cut and placed in test tubes containing 15 mL of distilled water for controls and 15 mL of each aqueous extract of diploid and mixoploid fenugreek plants for treatments. The tubes were incubated at room temperature for 24 h and 48 h and the initial electrical conductivity of the medium (EC-i) was measured using a digital conductivity meter (type BCT-4308). The samples were autoclaved at 121 °C for 20 min to release all electrolytes, cooled down to 25 °C and the final electrical conductivity (EC2) measured.

The electrolyte leakage (EL) was calculated according to the following formula (Lutts et al., 1996):

EL = (EC1/EC2) x 100.

2.4. Lipid peroxydation

Frozen samples (200 mg) (root and leaves) were homogenized with a mortar kept on ice and thoroughly mixed with 2.5 mL of 67 mM

phosphate buffer (pH = 7) and 0.05 g PVP, which adsorbs polyphenols. After centrifugation (2000 g for 15 min at 4 °C), the supernatant was used to determine lipid peroxidation (Doblinski et al., 2003). A 750 |jL of enzyme extract was added to 3 mL of 0.5% TBA (prepared in 20% TCA). The homogenate was incubated at 90 °C for 10 min. The reaction was stopped quickly by cooling the mixture in ice. Then, the mixture was centrifuged and the supernatant absorbance was measured at 532 and 600 nm, and the MDA concentration was calculated using the extinction coefficient of 155 mM-1 cm-1 (Doblinski et al., 2003).

2.5. Cell metabolic activity

The fresh plant material (100 mg) of germinated seeds or seedlings of lettuce grown in the absence or the presence of fenugreek extracts was washed and dried quickly between blotting paper, then incubated in 5 mL of TTC (0.2%, pH = 7) at 37 °C for 4 h in the dark. The reaction was stopped by adding 0.5 mL of sulfuric acid (1 M). Thereafter, the plant material was removed, washed with distilled water, dried quickly between filter paper and ground in a mortar placed in ice containing 3.5 mL of ethyl acetate. The homogenate was filtered through a paper Whattman No. 1 and the volume was adjusted to 7 mL with ethyl acetate. The absorbance was measured at 485 nm and the amount of formazan was calculated as follows (Sampietro et al., 2006):

Formazan content(%) = DO485 treatment/DO485 control.

2.6. Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) activities

Extraction and assay of enzymes were prepared under optimal conditions of pH and temperature. The fresh plant material (1 g) was ground in a mortar placed in ice (5 °C) containing 20 mL of borate buffer (0.1 M, pH = 8.7). The homogenate was filtered through filter paper Whattman No. 1. After centrifugation at 15,000 tr/min at 5 °C for 10 min, the supernatant recovered constituted the crude enzyme extract.

The PAL activity was determined according to Takayoshi and Kawamura (1964). The initial optical density (DO^ of the reaction mixture (1 mL) containing 50 mM l-phenylalanine and 0.2 mL of the crude enzyme extract was determined at 270 nm. After incubation at 40 °C for 90 min, the reaction was stopped by placing the tubes in ice, and the staining intensity was determined at 270 nm (DO2).

For the TAL, the fresh plant material (1 g) was ground in a mortar placed in ice (5 °C) containing 20 mL of borate buffer (0.1 M, pH = 9). The homogenate was filtered through filter paper Whattman No. 1. After centrifugation at 15,000 tr/min at 5 °C for 10 min, the supernatant recovered constituted the crude enzyme extract.

The TAL activity was performed according to Takayoshi and Kawamura (1964). The initial optical density (DO1) of the reaction mixture (1 mL) containing 10 mM l-tyrosine and 0.3 mL of crude enzyme extract was determined at 333 nm. After incubation at 40 °C for 90 min, the reaction was stopped by placing the tubes in ice and staining intensity was determined at 333 nm (DO2).

2.7. Proline content

Proline in lettuce roots and leaves was extracted and analyzed according to Bates et al. (1973). Ten milligram (10 mg) of dry plant material was mixed with 1.5 mL aqueous sulfosalicylic acid (3%, w/v). The homogenate was centrifuged at 13,000 tr/min for 10 min, and the supernatant was transferred to a fresh 1.5 mL tube. The extracted solution was reacted with an equal volume of glacial acetic acid and ninhydrin reagent (1.25 g ninhydrin in 30 mL glacial acetic and 20 mL 6 M H3PO4) and incubated at 100 °C for 1 h. The reaction was terminated by placing the tube in an ice bath. The reaction mixture was vigorously

mixed with 2 mL of toluene. After warming at 25 °C, the chromophore was measured at 520 nm. l-Proline was used as a standard.

2.8. Chlorophyll and carotenoid content

The contents of chlorophylls a and b and carotenoids were determined according to a method slightly modified from Lichtenthaler and Wellburn (1983). Two leaves of about 100 mg fresh weight were placed in 5 mL of 80% acetone. After filtration, the absorbance was carried out at 663, 645 nm and 440 nm. The pigment levels, expressed as mg/g fresh weight, were calculated from the following equations:

Total chlorophyll (mg/g) = 20.2 A645 + 8.02 A663 Chlorophyll a(mg/g) = 12.7 A663-2.69 A45

Chlorophyll b(mg/g) = 22.9 AM5-4.68 Ag63

Carotenoids (mg/g) = (4.7 A440-(1.38 A663 + 5.48 A645)

2.9. Secondary metabolites production in lettuce

Lettuce seedlings grown in the presence and the absence of fenugreek extracts harvested and frozen (separating roots and leaves) were homogenized in 80% methanol for 24 h. The homogenate was cen-trifuged at 10,000 g for 20 min at 4 °C and the supernatant was used to determine the levels of secondary metabolites (García-Sánchez et al., 2012).

2.9.1. Total phenolic (TP) content

The TP content were measured using the modified Folin-Ciocalteau method (Velioglu etal., 1998). Sample extract (100 |jL) was mixed with 500 |jL of 1/10 diluted (in Milli-Q water) Folin-Ciocalteau phenol reagent and allowed to react for 5 min in the dark at room temperature. Then 400 |aL of sodium bicarbonate (7.5%) were added to the mixture. After 90 min of incubation in the dark at 30 °C, the absorbance was read at 765 nm. TP content were expressed as mg gallic acid equivalent/g dry matter (mg GAE/g dw) using gallic acid calibration curve (R2 = 0.971).

2.9.2. Total flavonoid (TFd) content

The TFd content was determined spectrophotometrically according to standard method (Quettier et al., 2000). Briefly, 0.5 mLof2% solution of AlCl3 in methanol was mixed with the same volume of extract. Absorption readings at 430 nm were taken after 30 min against a blank. TFd content was expressed as mg quercetine equivalent/g dry weight (mg QE/g dw) using quercetine calibration curve (R2 = 0.997).

2.9.3. Total precipitable alkaloid (TA) content

The TA content was determined by spectrophotometric method with Dragendorff reagent (Stumpf, 1984). Principally, 300 ^Lof plant extract was mixed with 100 |jL of Dragendorff reagent. After centrifuga-tion at 7000 g for 1 min, the supernatant was removed and dissolved in 1 mL of 2.45 M Nal. An aliquot of 10 ^L of each tube was added to 1 mL of 0.49 M Nal, after which the absorbance was read at 467 nm. TA content was expressed as mg papaverine hydrochloride equivalent/g dry weight (mg PAHE/g dw) using papaverine hydrochloride calibration curve (R2 = 0.998).

2.10. Statistical analysis

All data were reported as mean ± standard deviation (S.D.) of five replicates and analyzed using the program PASW Statistics 18.

Differences between the means were established using general linear model (GLM) procedure (p < 0.05) related to the two variables: extraction type and phenological stage. Differences at the 5% level (p < 0.05) were considered statistically significant.

3. Results and discussion

3.1. Effect of fenugreek aqueous extracts on lettuce germination

3.1.1. Electrolyte leakage by lettuce germinated seeds

Table 1 shows the variation of electrolyte leakage from lettuce seeds after immersion in different aqueous extracts of fenugreek prepared at concentration inducing a 50% germination rate (IC50). Electrolyte leakage varied with the incubation time of seeds and the extract origin (developmental stage and ploidy level of fenugreek). Thus, extracts of the two types of plants harvested at flowering stage did not have a significant effect on this parameter, and electrolyte leakage did not exceed 12.86% after 48 hours immersion. Those of the biomass collected at the two other stages were more harmful, especially that of mixoploid at the fruiting stage, which led to a loss of 193.5% of electrolytes, compared to the control, after 24 h, and 272.79% after 48 h. The extract of the diploid plants, at the same stage, was responsible for a respective leak of 26% and 8%. At vegetative stage, diploid plant extract was slightly more harmful with a leak of 40.52% and 66.35% after 24 and 48 h of incubation, respectively. These values were 30.81% and 47% in the presence of the mixoploid plants extract (Table 1).

The leakage of electrolytes under the influence of aqueous extracts reflects a disruption of membrane permeability and thus its integrity and can lead to cell death. This parameter is often used as an indicator of damage to the plasma membrane. According to Bogatek et al. (2005), the increase of electrolyte leakage during germination may indicate an inability to maintain the consistency of membranes, ultimately resulting in the perturbation of germination (Bogatek et al., 2005). The effects of plant extracts on this parameter are reported in some studies. Thus, Krystyna et al. (2007) and Bogatek et al. (2005) showed that the treatment of mustard seeds (Sinapis alba L.) with aqueous extract of leaves of sunflower (Helianthus annuus L.) harvested at flowering led to a rapid and significant increase in the leakage electrolytes, resulting in damage to the plasma membrane.

3.1.2. Lipid peroxidation in lettuce germinated seeds

Polyunsaturated fatty acids are the major lipid component of

the membrane susceptible to peroxidation and degradation. Indeed, the increase in membrane permeability under allelochemicals stress corresponds to an increase in lipid peroxidation estimated by malondialdehyde (MDA) accumulation. This assay was performed on treated and untreated seeds by aqueous extracts. The results showed no significant differences between the effects of different fenugreek extracts and control, except the extract of mixoploid plants harvested at fruiting stage which induced an increase of 14 times in the MDA content of lettuce seeds, compared to the control (Table 1).

Malondialdehyde (MDA) is considered a sensitive marker commonly used to assess the lipid peroxidation of the membrane (Goel and Sheoran, 2003). Allelochemicals could eventually damage cell membranes through direct interaction with a constituent of the membrane or as a result of an impairment of some metabolic function necessary to the maintenance of membrane function (Rice, 1984). The increase in lipid peroxidation is also a marker of oxidative stress (Schopfer et al., 2001) and is used as a possible explanation of lipid peroxidation during germination (Schopfer et al., 2001). Indeed, it is reported that the MDA generates additional free radicals (Mustafa, 1990). Bogatek et al. (2006) showed that the decrease in germination capacity of mustard seed was correlated with the high level of lipid peroxidation (estimated by the content of MDA) caused by the extracts of two culti-vars of sunflower. Similarly, Krystyna et al. (2007) reported that the content of MDA of germinated mustard seeds in the presence of

Electrolyte leakage (%) of lettuce pre-germinated seeds, malondialdehyde content (MDA) (% of control) and formazan content (% of control) of lettuce germinated seeds in the presence of aqueous extracts (concentration inducing a reduction 50% of germination) of T.foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage.

Vegetative

Flowering

Fruiting

Diploid

Mixoploid

Diploid

Mixoploid

Diploid

Mixoploid

Electrolytes leakage 24 h 48 h

Lipid peroxidation (MDA) Formazan content

40.52b ± 5.23 66.35b ± 5.14 105.36a ± 10.27 55.99a ± 7.68

30.81b ± 9.53 47.07b ± 9.85 89.77a ± 8.82 52.84a ± 10.85

6.80a ±1.13 10.86a ± 1.25 44.03a ± 6.62 76.01b ± 9.94

1.41a ± 0.85 12.86a ± 4.56 79.82a ± 5.19 68.35b ± 6.71

25.79b ± 8.69 8.44a ± 1.50 131.27a ± 18.54 78.06b ± 6.44

193.51c ± 11.89 272.79c ± 12.36 1360.79b ± 30.21 71.95ab ± 4.77

All analyses are the mean of five measurements ± standard deviation. Means followed by at least one same letter are not significantly different at p < 0.05.

sunflower extract increased until the fourth day and remained at a constant value until the end of the experiment. Furthermore, the content of MDA increased significantly in germinated grain of Tritiaim aestivum L. and Brassica napus L. in the presence of the crude methanol extract of the roots and aerial parts of Phytolacca latbenia (Nazif et al., 2013).

3.1.3. Cell metabolic activity in lettuce germinated seeds

Table 1 reports the formazan content, expressed as a percentage of the control in lettuce seeds treated with different fenugreek extracts. These contents reflect the metabolic activity of cells, mainly the activities of dehydrogenase enzymes and thus mitochondrial respiration. The results showed a more or less significant decrease of formazan production under the effect of different extracts compared to the control. This content was 55.99, 76 and 78% in seeds subjected to the effect of extracts of diploid plants harvested at the vegetative, flowering and fruiting stage, respectively. These values were 52.84, 68.35 and 71.95% in the presence of the mixoploid plants extracts (Table 1).

Cellular respiration is a vital phenomenon during germination, providing a supply of ATP to the embryo allowing it to resume its metabolic activities. Reducing breathing seeds subjected to the action of plant extracts could explain the arrest of germination in their presence. The effect of plant extracts or allelochemicals on breathing is reported in the literature (Sampietro et al., 2006; Rashid et al., 2010). The decrease in the activity of dehydrogenases could be a reflection of cell damage due to exposure to allelochemicals present in extracts of fenugreek. Indeed, the lower production of formazan was recorded in the presence of extracts from the two types of plants collected at the vegetative stage, where mitochondrial respiration appears to be reduced by almost half.

Furthermore, our results showed that membrane damage of lettuce seeds were affected differently depending on the origin of extract. For biomass harvested at the vegetative stage, the diploid was more harmful, although the difference is not very significant, whereas the fruiting plant material of mixoploid was much more harmful. This reflects, and

could be related with a change in the chemical composition of the aqueous extracts (Omezzine and Haouala, 2013) and probably the excessive accumulation responsible for membrane damage by mixoploid plants during its development substance. Indeed, Omezzine et al. (2014) showed that the composition of mixoploid biomass collected at fruiting stage is enriched with two flavonol glycosides: kaempferol 3-O-p-d-glucopyranoside and quercetin-3-glucoside galactoside, compared to diploid. This allows speculating that these flavonoids may be responsible for the inhibition of lettuce germination (Omezzine et al., 2014) by inducing damage of the cell membrane.

3.2. Effect of fenugreek aqueous extracts on lettuce seedlings growth

32.1. Electrolyte leakage by lettuce roots

The electrolyte leakage from lettuce roots was determined by measuring the conductivity of the medium where they were immersed (Fig. 1). The results showed a significant leakage of electrolytes in the presence of different extracts which varied with the origin of extract and increased with the incubation time. Indeed, after 24 h of incubation, extracts of diploid plants harvested at vegetative, flowering and fruiting stages induced leakage of44,104 and 23%, respectively, against 30,47 and 41% in the presence of extracts from mixoploid plants. After 48 h, the leakage of electrolytes has strongly increased, especially in the presence of extracts from diploid plants harvested at the vegetative and flowering stages with respective values of 305% and 475%, against 256% and 123% in the presence of those of mixoploid plants. The extracts of the two types of plants collected at fruiting stage induced an average leakage of 134.5% (Fig. 1).

Damage of the cell membrane is almost ubiquitous due to a variety of stress, and loss of membrane integrity response is among the main factors that determine cell injury. Indeed, Kaur et al. (2010) reported that the change in membrane permeability affects all other physiological and biochemical processes related to the operation of the

Fig. 1. Electrolyte leakage (%) of lettuce roots grown on control medium, after an immersion of 24 and 48 h in distilled water or aqueous extracts (at IC50 for root growth) of T.foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

membrane. Tanatson et al. (2013) showed that the increase in electrolyte leakage from the roots of barnyardgrass (Echinochloa crus-galli), under the effect of essential oils of Cymbopogon citratus, is the result of a disruption of the membrane integrity which increase its permeability. Singh et al. (2005) and Kaur et al. (2010) reported that some terpenes inhibit plant growth as a result of electrolyte leakage. Indeed, helvolic acid (a terpenoid) has increased the electrolyte leakage by a rice cultivar (Sakthivel et al., 2002). Various other steroidal compounds are reported to have increased the electrolyte leakage by many weeds and crops of different tissues (Hoagland et al., 1996). According to Latkowska et al. (2008), treatment of roots of tomato with (+)-usnic acid increased the electrolyte leakage by altering the permeability of the plasma membrane reflecting a disturbance of the structure.

3.2.2. Lipid peroxidation in lettuce seedlings

Fig. 2 shows the MDA contents in lettuce roots and leaves, grown in the presence of different fenugreek extracts, expressed as a percent of the control. Overall, the results show a greater sensitivity of the root cell membranes to allelochemicals stress compared to that of leaves.

For roots, the MDA content increased significantly, 4.8; 6 and 2 times compared to the control, in the presence of extracts of diploid material harvested at vegetative, flowering and fruiting stage, respectively. These factors were 5.8; 2.23 and 2.37 times in the presence of mixoploid plant extracts (Fig. 2). For leaves, the highest content was recorded in the presence of extracts of material collected at the vegetative stage for the two types of plants with respective stimulation of 4.35 and 6.58 times. The MDA content increased by half (1.5 times) in the presence of extracts of the two types of plants collected at flowering, while it almost doubled (2.27 times) under the effect of the extract of diploid plants harvested at fruiting stage and was comparable to the control (1.07 times) under the mixoploid extract (Fig. 2).

The MDA accumulation due to lipid peroxidation has been reported in response to a variety of abiotic and biotic stresses (Apel and Hirt, 2004). As noted above, the lipid peroxidation of membrane and membrane damage is a common indicator of allelochemicals stress (Rice, 1984; Singh et al., 2006). An accumulation of MDA was recorded, with a largest content in lettuce roots compared to leaves, this result is consistent with that of Batish et al. (2006) who reported higher levels of MDA in the roots of Phaseolus aureus under the effect of 2-benzoxazolinone. This result indicates a greater impact of allelochemicals on the roots, resulting in a greater production of ROS and thus a higher level of oxidative stress. The effect of some allelochemicals on membrane lipid peroxidation is reported in the literature, thus, 3,4-dihydroxy phenylalanine (l-DOPA), a powerful phytotoxin ofMucuna increased the MDA levels and caused oxidative damage in the roots of lettuce (Hachinohe and Matsumoto, 2005). Studies on cucumber and sorghum roots showed

that the disintegration of the membrane under the effect of allelopathic compounds is proportional to the lipid peroxidation measured by MDA content (Zeng et al., 2001). In addition, maize roots treated for 2 h with a solution of 0.5 mM ferulic acid showed higher by 22%, compared to control, levels of MDA, and a 36% increase in response to the same concentration of p-coumaric acid (Gmerek and Politycka, 2011). The 2-benzoxazolinone (BOA) has also increased, significantly, the MDA content in the roots and leaves of P. aureus (Batish et al., 2006). A similar result is obtained with the leaves of Brassica oleracea var. capitata cultured in the presence of cinnamic acid at various concentrations (Singh et al., 2013).

3.2.3. Variation of phenylalanine ammonia-lyase (PAL) activity in lettuce seedlings

The PAL activity was measured in lettuce leaves and roots grown in the absence and the presence of different aqueous extracts of fenugreek diploid and mixoploid plants harvested at three stages (Fig. 3). The effect of different extracts was highly significant, whether in the two organs (leaf and root) or in relation to their origin (ploidy level and developmental stage).

For roots, extracts of diploid plants harvested at the vegetative stage have significantly improved the PAL activity (0.032 x 10-5 ^mol/min g MF), which has almost doubled compared to the control (0.018 10-5 ^mol/min g MF). However, in the presence of extracts from the two other materials, this activity has decreased on average by half (Fig. 3). In the presence of extracts of mixoploids, the activity of this enzyme was reduced compared to the control in all cases, with lower activity recorded in the presence of extracts from material collected at flowering stage where the reduction was 86%, in the other two cases an average decrease of 22% was noted (Fig. 3).

For leaves, in the presence of aqueous extracts of diploid plants collected at flowering, the activity of this enzyme has been improved by 1.52 times, and was close to the control in the two others cases. In the presence of mixoploid extracts, PAL activity was stimulated by the extract of material collected at the vegetative stage (1.8 times), reduced 75% by the extract of the second stage and slightly improved (1.3 times) in the third case (Fig. 3).

The PAL is a key enzyme in the phenylpropanoid pathway and the increase in its activity was correlated with an increased production of phenylpropanoid (Ozeki and Komamine, 1985). It converts l-phenylalanine to trans-cinnamic acid, which is the precursor for the synthesis of most of the phenolic compounds such as lignin and salicylic acid (Nugroho et al., 2001). These compounds are involved in building pecto-cellulosic walls (Chen and Mc Clure, 2000). PAL is generally considered a marker of environmental stress in different plant species (Mac Donald and D'Cunha, 2007) and the variation of its activity is a response

Fig. 2. Content of malondialdehyde (MDA) (%) of lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at 1C50 for root growth) of T. foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

Fig. 3. Activity of phenylalanine ammonia-lyase (PAL) (% of control) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T.foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

to various biotic and abiotic stresses (Camacho-Cristabal et al., 2002). Some studies have shown that allelochemicals such as ferulic acid and p-coumaric acid have increased significantly the activity of the PAL (dosSantos et al., 2004). We recorded a variation in this enzyme activity in lettuce roots and leaves, under fenugreek extracts, which is consistent with the literature. Thus, Bellini and Van Poucke (1970) showed that the PAL activity was higher in the roots of radish compared to control hypo-cotyls, contrary to our result. Furthermore, the increased activity of PAL was attached to the inhibition of root growth of maize (Devi and Prasad, 1996), cucumber (Politycka, 1998) and soybean (Herrig et al., 2002), when these species are exposed to derivatives of cinnamic and benzoic acids. Similarly, Reid and Marsh (1969) showed that the activity of PAL was enhanced by the application of gibberellic acid to corn seedlings. The results for the change in the activity of this enzyme are more or less controversial and Shann and Blum (1987) showed that the activity of PAL was not affected by ferulic acid in the roots of cucumber. However, Politycka (1998) showed that the same acid has increased its activity, while reducing root growth of cucumber.

3.2.4. Variation of tyrosine ammonia lyase (TAL) activity in lettuce seedlings

The activity of TAL, expressed in ^mol/g min MF, in the roots and leaves of the lettuce grown in the presence of different aqueous extracts of diploid and mixoploid fenugreek, varied with three developmental stages. The activity of this enzyme has not strongly varied in the two target organs (Fig. 4).

Indeed, in the presence of aqueous extracts from diploid plants, the activity of the TAL did not show significant differences, in lettuce roots, compared to the control (0.013 • 10-5 ^mol/min g MF against an average of 0.014 • 10-5 ^mol/min g MF). However, extracts from mixoploid plants harvested at flowering and fruiting stages have improved its activity by an average of 27%, while the extract of harvested plants at the vegetative stage had no effect (Fig. 4).

In lettuce leaves, the TAL activity was stimulated by an average of 28% in the presence of extracts from diploids harvested at the flowering and fruiting stages and was comparable to the control in the third case (Fig. 4). Regarding mixoploid materials, the extract of vegetative stage had no effect, while the two others have slightly inhibited the TAL activity compared to control by an average of 11.5% (Fig. 4).

Thus, in the roots, the highest activity of the TAL was observed in the presence of extracts from mixoploid plants harvested at the last two stages of development, whereas for lettuce leaves, the same result was obtained with extracts from the diploid.

The TAL converts l-tyrosine to ammonia and p-coumaric (Neish, 1961). This enzyme is a member of a family of ammonia-lyases that deaminate the aromatic amino acids, l-His, l-Phe, and l-Tyr (Poppe and Rétey, 2005). It has been studied less than the PAL, and it remains unclear whether TAL activity is due to a capability of PAL to accept tyrosine as a substrate or due to the activity of a specific enzyme. Reid et al. (1972) showed that the application of gibberellic acid to maize seedlings resulted in an increase in the activity of the TAL. Similarly, Teresa et al. (1998) reported an improvement in its activity in young plants

Fig. 4. Activity of tyrosine ammonia-lyase (TAL) (% of control) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T.foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

Fig. 5. Formazan content (% of control) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T.foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

of Fragaria ananassa treated by gibberellic acid (30 and 60 ^g/L). However, Kazuyuki et al. (1997) reported that the application of abscisic acid (ABA) in roots of wheat (T. aestivum L.) resulted in a decrease in the activity of this enzyme. The high level of phenylalanine and tyrosine may also be caused by the diversion of intermediates of glycolysis and photosynthesis to the shikimate pathway, suggesting a link between the allelopathic stress and lignification (Dos Santos et al., 2008). The shi-kimic acid pathway (which leads to the synthesis of aromatic amino acids such as phenylalanine and tyrosine) and the phenylpropanoid pathway (which leads to the synthesis of lignin) are significantly related (Wildermurth, 2006). This indicates that any effect (biotic or abiotic stress) of these enzymes can alter the flow of metabolites in these pathways, and therefore affect the metabolism and plant development.

3.2.5. Cell metabolic activity in lettuce seedlings

The formazan content, expressed as a percent of control, in lettuce roots and leaves grown in the presence of different extracts of fenugreek diploid and mixoploid is shown in Fig. 5. The results showed a high variability depending on the developmental stage and level of plant ploidy.

For the roots, the formazan level was greatly reduced compared to the control in all cases. In the presence of extracts from diploid plants, the highest content was recorded in the presence of plant material harvested at flowering (43%) followed by the fruiting stage (33%) and the vegetative one (10%). In the presence of mixoploid extracts these levels were 30%, 11% and 43%, respectively (Fig. 5). The formazan level was higher in lettuce leaves indicating greater metabolic activity. As for the effect of different extracts, in the presence of diploid plants, the

contents of formazan were 66%, 66% and 82% in presence of extracts corresponding to the vegetative, flowering and fruiting mixoploids; these values were 63%, 90% and 50% in the presence of mixoploid extracts (Fig. 5).

The reduction in TTC (2,3,5-triphenyl tetrazolium chloride) is an indicator of the activity of mitochondrial dehydrogenases, and it is often used for the measurements of cellular respiration (Musser and Oseroff, 1994; Stowe et al., 1995). Rashid et al. (2010) have shown that extracts from the leaves and roots of Pueraria montana caused a significant decrease in the production of formazan in the roots of lettuce and radish. In addition, phenolic acids (isolated from leachate of Saccharum officinarum) have reduced the rate formazan in the roots of lettuce (Sampietro et al., 2006). These authors associate the decrease in the activity of dehydrogenases to a decrease in ATP production, resulting in the inhibition of root growth.

3.2.6. Accumulation ofproline in lettuce seedlings

Fig. 6 shows the levels of proline in the roots and leaves of lettuce grown under the same conditions as previously. On control medium leaves showed a higher amount (0.16 ^mol/g DM) than roots (0.10 ^mol/g DM).

Proline accumulation in roots was similar to the control in the presence of extracts of diploids harvested at the first two stages and was reduced by half with extract of the third stage. In the presence of diploid extracts, proline content increased 1.61 times under the effect of the extract of plants collected at fruiting stage, was similar to the control with vegetative material and reduced by 0.64 times with that of the third case (Fig. 6).

Fig. 6. Proline content (|jmol/g DW) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T.foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

Control Vegetative Flowering Fruiting Control Vegetative Flowering Fruiting Control Vegetative Flowering Fniitin:

□ Diploid oMixoploid □ Control Developmental Stage

Fig. 7. Chlorophyll content (mg/g FW) in lettuce leaves grown on control medium or medium added with different aqueous extracts (atIC50 for root growth) of T. foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

In the leaves, proline accumulation increased in all cases except in the presence of extract material corresponding to fruiting stage of mixoploids, where the content was comparable to the control. In the presence of extracts from diploids harvested at vegetative, flowering and fruiting stages, the increase was 1.52,1.39 and 1.29 times respectively. In the presence of the first two extracts of mixoploids an average stimulation of 1.44 times was recorded (Fig. 6).

The accumulation of proline in plants is an indication of a disturbed physiological state, triggered by biotic or abiotic stress. Determining its rate is a useful assay to monitor and evaluate the physiological tolerance of plants to stress (Abraham et al., 2010). Proline acts as an electron acceptor and prevents membrane damage (Ain-Lhout et al., 2001). It also provides protection against photosynthetic perturbations induced by ROS (Hare et al., 1998). In this study, an accumulation of this metabolite was noted in some cases, stimulating factors ranging between 1.37 and 1.61 times. Similar results have been reported in the literature. So Djanaguiraman et al. (2005) showed that allelochemicals present in the leachate of eucalyptus leaves have increased the proline content in sorghum and mung bean. This content has increased in the roots of Cicer arietinum treated by a-pinene 2.5 mM and 5 mM, of 1.3 and 1.9 times respectively compared to the control (Singh et al., 2006). Similarly, Thapar and Singh (2006) noted a stimulation of proline production in the leaves of Parthenium hysterophorus treated by leachate leaves of Cassia tora; this could be due to the induction of specific proteins in response to oxidative damage caused by allelochemicals stress (Mishra et al., 2006). In addition, the proline content, recorded in this study, in the lettuce leaves was almost double than that of roots. A similar result was reported by Latkowska et al. (2008) who reported that proline content was approximately two times higher in the leaves compared to that of the roots of tomato (Lycopersicon esculentum Mill.) treated by ( + ) usnic acid.

3.2.7. Photosynthetic pigment content

3.2.7.1. Chlorophyll content. The contents of chlorophylls a (chl a) b (chl b) and total (chl t) were determined in lettuce leaves collected at the end of culture (Fig. 7). The results showed a slight variation depending on the developmental stage of fenugreek, but no change with their ploidy level was observed. Indeed, chl a content was comparable to the control in all cases, while a slight decrease was recorded in chl t in the presence of extracts of both plants harvested at vegetative and flowering stage. This decrease was due to a reduction in the rate of chl b, recorded under the same conditions, with respective reduction factors of 0.69 and 0.81 times. The extracts of the biomass collected at fruiting stage had no effect (Fig. 7).

Chlorophylls are the basic elements of pigment-protein complexes incorporated in photosynthetic membranes and play a major role in photosynthesis. The chlorophyll content can provide insight on the

mode of action of allelochemicals present in the environment (Kirby and Sheahan, 1994). However, the decrease in chlorophyll content per unit of plant material does not necessarily mean the inhibition of growth and vice versa (Verdisson et al., 2001). In the present study, a slight reduction in the content chl t was recorded, consistent with a decrease of chl b. The allelopathic effect of stress on the chlorophyll content has been reported in the literature. Thus, Patterson (1981) found that treatment of soybean plants by ferulic, p-coumaric and vanillic acid has significantly reduced the chlorophyll content. Similarly, the content of chl a fell under the influence of phenolic acids in rice (Yang et al., 2002)), secalonic acid in sorghum (Zeng et al., 2001), and monoterpenes in Cassia occidentalis (Singh et al., 2002). Siddiqui (2007) reported a reduction in chlorophyll content of Vigna mungo due to the presence of allelochemicals in the leachate of black pepper. Rice (1984) suggested that some allelochemicals can interfere with the synthesis of porphyrin precursor of chlorophyll biosynthesis. Under the pressure of allelochemicals stress, the reduction of photosynthetic pigments was reported and attributed to their biosynthesis inhibition (Singh et al., 2009) or to stimulation of their degradation (Yang et al., 2004).

3.2.7.2. Carotenoid content. The carotenoid contents decreased significantly, compared with the control in the presence of all fenugreek extracts. Thus, reduction factors of 0.8, 0.66 and 0.45 times, were recorded in the presence of extracts from diploid plants harvested at vegetative, flowering and fruiting stage respectively. In the presence of extracts from mixoploids, these factors were 0.69; 0.56 and 0.48, respectively (Fig. 8).

Fig. 8. Content in carotenoids (mg/g FW) in lettuce leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T. foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

Fig. 9. Total phenolic contents (mg GA/g FW) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T. foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

Unlike the content of chlorophylls, carotenoid content has been reduced as a result of allelochemical stress. The contribution of this type of pigment in photosynthesis is well elucidated and it is reported that the decrease may be the result of either an alteration of the carotenoid biosynthesis or inhibition of the enzyme protoporphyrinogen oxidase leads in chlorophyll biosynthesis (Yang et al., 2002). Carotenoids are antioxidant (Mishra et al., 2006) and their content can be influenced, positively or negatively, as a result of allelochemical stress. Indeed, Darier and Tammam (2012) have shown that aqueous extracts of the aerial parts of Achillea santolina have increased the carotenoid content in Viciafaba and Hordeum vulgare.

3.2.8. Accumulation of secondary metabolites in lettuce seedlings

3.2.8.1. Total phenolic (TP) content. For roots, TP levels have increased compared to the control, by 14 times on average, in the presence of extracts from diploids harvested at vegetative and flowering stages and by 7 times in the third case. Under the effect of mixoploid extracts, these factors were respectively 12, 6 and 21 times (Fig. 9).

For lettuce leaves, the TP contents were multiplied by 1.86 times under the effect of diploid extracts harvested at flowering stage, in the other two cases they were similar to the control. However, in the presence of extracts of mixoploids, the TP contents decreased compared to control, especially in the presence of extracts from the material collected at flowering and fruiting stages where a respective reduction of 60% and 73% was noted; in the other case the content was close to the control (Fig. 9).

3.2.8.2. Total flavonoid (TFd) content. In the presence of extracts of both types of plants harvested at the vegetative, flowering and fruiting stage, TFd root content was increased by, respectively, 2.15, 1.8 and 1.4 times, compared to the control (Fig. 10). For leaves, the effect of different extracts was depressive on the TFd accumulation, with no significant difference between the extracts of different development stages but a significant effect was recorded with ploidy level. Thus, under the effect of diploids extracts a reduction of 0.87 times, on average, was recorded and 0.72 in the case of mixoploid extracts (Fig. 10).

3.2.8.3. Total precipitable alkaloid (TA) content. Fig. 11 shows thatTA content varied with ploidy level of plants, the harvest stage but also the target organ. For lettuce roots, TA content was not affected in the presence of extracts of diploids harvested at the first two stages, and was reduced by 0.73 in the third case. However, under the effect of extracts of mixoploids, this content has been increased by 1.34,1.64 and 2.18 times, when they correspond to the material collected at the vegetative, flowering and fruiting stage, respectively (Fig. 11).

Under control condition, the TA content of lettuce leaves, was almost twice that of roots. However, all extracts of diploids have halved this content, regardless of the harvest stage. This reduction was recorded only in the presence of the extract of the vegetative stage of mixoploid; no significant effect was recorded in the other two cases (Fig .11).

Secondary metabolites play a major role in the adaptation of plants to environmental conditions and the circumvention of stress conditions. Indeed, it has been reported that accumulation often occurs in plants subjected to various biotic and abiotic stresses (Camacho-Cristabal

Fig. 10. Total flavonoid contents (mg QE/g FW) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T. foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

Fig. 11. Total precipitable alkaloids (mg PAHE/g FW) in lettuce roots and leaves grown on control medium or medium added with different aqueous extracts (at IC50 for root growth) of T. foenum-graecum aerial parts (diploid and mixoploid) harvested at the vegetative, flowering and fruiting stage. The bars on each column show standard error. Value = average ± S.E., n = 5. Different letters on columns indicate significant differences among concentrations at p < 0.05.

et al., 2002; El-Rokeik, 2007). Although little information is available on the relationship between allelochemical stress and secondary metabolites (Zou et al., 2006), numerous studies have shown that plants could improve the synthesis of phenolic substances to overcome the stress induced by allelochemicals as phenolic compounds work, among others, such as antioxidants (Herrig et al., 2002). Thus, the content of phenolic compounds increased in wheat seedlings due to the allelopathic effect of Ranunculus arvensis (Bansal, 1997). The inhibitory effects of eucalyptus on weeds are positively correlated with the accumulation of total phenols, compared to their controls (El-Rokiek and Eid, 2009). Allelochemicals present in the leachate of eucalyptus leaves have increased the phenol content in sorghum and beans (Djanaguiraman et al., 2005). And finally, the content of these compounds increased significantly in the roots of watermelon (Citrullus lanatus) cultivated in a nutrient solution containing extracts from the same plant (Zou et al., 2006). Some researchers have reported that allelochemicals have inhibitory effects on physiological processes that result in a reduction in growth (Jefferson and Pennacchio, 2003).

The disruption and inhibition of lettuce growth recorded in previous works (Omezzine and Haouala, 2013; Omezzine et al., 2014), in the presence of different extracts of fenugreek (diploid and mixoploid) could be attributed to the increase of electrolyte leakage, the MDA accumulation and the mitochondrial respiration disruption (resulting in the decrease in the supply of ATP required for the processes demanding) in lettuce roots and leaves. Indeed, damage of cell membrane was significantly marked in lettuce roots, especially under the influence of extracts of diploid plants harvested at the flowering stage and those mixoploid plants harvested at vegetative stage, which were the most vulnerable. The greatest effect of diploid extracts, particularly in the flowering stages, could be attributed to their greater wealth in total polyphenols; however, the high effect of mixoploid extract (at vegetative stage) could be attributed to its richness in total flavonoids (Omezzine and Haouala, 2013; Omezzine et al., 2014). The reduction in chlorophyll content does not seem to explain the reduction in seedling growth, hence it is not very important and the chl content was not affected. The decrease in carotenoid content may explain, in part, the decrease in the growth of lettuce plants already registered (Omezzine and Haouala, 2013; Omezzine et al., 2014). Note that the effect of the two plant extracts was comparable and those of the fruiting stage were the most harmful.

4. Conclusion

This study showed that allelochemicals present in the aqueous extracts of diploid and mixoploid fenugreek caused a degradation of the plasma membrane in lettuce germinated seeds and roots, evidenced by a strong electrolyte leakage and increased of lipid peroxidation. In addition, these allelochemicals have decreased mitochondrial

respiration in seeds, roots and leaves, as well as the pigment content. All these disturbances are responsible for the lettuce seedling reduction growth, registered in the presence of aqueous extracts. Moreover, lettuce seedlings have circumvented this stress by setting up a defense strategy that involves an increase in some metabolite production, such as proline, polyphenols, flavonoids and alkaloids. This metabolite accumulation is the result of an increase in the lyase activity, recorded in some cases, reflecting the implementation of this strategy. The mixoploidization seems to be a simple and effective biotechnology tool to improve (in quantity and quality) the allelochemicals production, since the extract toxicity of diploid and mixoploid plants, was different. Hence, the aqueous extracts of mixoploid plants harvested at fruiting and vegetative stages were the most vulnerable and the first could be used as a pre-emergence herbicide and the second one as post emergence herbicide.

References

Abraham, E., Hourton-Cabassa, C., Erdei, L., Szabados, L., 2010. Methods for determination

of proline in plants. Methods Mol. Biol. 639,317-331. Abrahim, D., Braguini, W.L., Kelmer-Bracht, A.M., Ishii-Iwamoto, E.L., 2000. Effects of four monoterpenes on germination, primary root growth and mitochondrial respiration of maize. J. Chem. Ecol. 26, 611-624. Ain-Lhout, F., Zunzunegui, F.A., Diaz Barradas, M.C., Tirado, R., Clavijio, A., Garcia Novo, F., 2001. Comparison of proline accumulation in two Mediterranean shrubs subjected to natural and experimental water deficit. Plant Soil 230,175-183. Anaya, A.L., Pelayo-Benavides, H.R., 1997. Allelopathic potential of Mirabilis jalapa L. (Nictaginaceae): effect on germination, growth and cell division of some plants. Allelopathy J. 4, 57-68.

Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal

transduction. Annu. Rev. Plant Biol. 55, 373-399. Baar, J., Ozinga, W.A., Smeers, I.L., Kuyper, T.W., 1994. Stimulatory and inhibitory effects on needle litter and grass extracts on the growth of some ectomycorrhizal fungi. Soil Biol. Biochem. 26,1076-1079. Bansal, G.L., 1997. Allelopathic effect of buttercups on wheat varieties. Allelopathy J. 4, 139-142.

Barkosky, R.R., Butler, J.L., Einhellig, F.A., 2000. Caffeic acid-induced changes in plant water

relationships and photosynthesis in leafy spurge. J. Chem. Ecol. 26, 2095-2109. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-

stress studies. Plant Soil 39, 205-207. Batish, D.R., Singh, H.P., Setia, N., Kaur, S., Kohli, R.K., 2006.2-benzoxazolinone (BOA) induced oxidative stress, lipid peroxidation and changes in some antioxidant enzyme activities in mung bean (Phaseolus aureus). Plant Physiol. Biochem. 44,819-827. Baziramakenga, R., Simard, R.R., Leroux, G.D., 1994. Effects of benzoic cinnamic acids on growth, mineral composition and chlorophyll content of soybean. J. Chem. Ecol. 20, 2821-2833.

Bellini, E., Van Poucke, M., 1970. Distribution of phenylalanine ammonialyase in etiolated

and far-red irradiated radish seedlings. Planta 93, 60-70. Blum, U., 2005. Relationships between phenolic acid concentrations, transpiration, water utilization, leaf area expansion, and uptake of phenolic acids: nutrient culture studies. J. Chem. Ecol. 31,1907-1932. Bogatek R., Oracz, K., Gniazdowska, A., 2005. Ethylene and ABA production in germinating seeds during allelopathy stress. Proceedings of the 4th World Congress on Allelopathy, Australia. International Allelopathy Society. Bogatek R., Gniazdowska, A., Zakrzewska, W., Oracz, K., Gawronski, S.W., 2006. Allelopathic effects of sunflower extracts on mustard seed germination and seedling growth. Biol. Plant. 50,156-158.

Camacho-Cristábal, J.J., Anzelloti, D., González-Fontes, A., 2002. Changes in phenolic metabolism of tobacco plants during short-term boron deficiency. Plant Physiol. Biochem. 40,997-1002.

Chen, M., Mc Clure, J.W., 2000. Altered lignin composition in phenylalanine ammonia-lyase inhibited radish seedlings: implication for seedderived sinapoyl esters as lignin precursors. Phytochemistry 53,365-370.

Darier, S.M., Tammam, A.A., 2012. Potentially phytotoxic effect of aqueous extract of Achillea santolina induced oxidative stress on Vicia faba and Hordeum vulgare. Rom. J. Biol. 57, 3-25.

Devi, R.S., Prasad, M.N.V., 1996. Ferulic acid mediated changes in oxidative enzymes of maize seedlings: implications in growth. Biol. Plant. 38,387-395.

Djanaguiraman, M., Vaidyanathan, R., Annie Sheeba, J., Durga Devi, D., Bangarusamy, U., 2005. Physiological responses of Eucalyptus globules leaf leachate on seedling physiology of rice, sorghum and blackgram. Int. J. Agric. Biol. 7,35-38.

Doblinski, P.M.F., Ferrarese, M.L.L., Huber, DA, Scapim, C.A., Braccini, A.L., Ferrarese-Filho, O., 2003. Peroxidase and lipid peroxidation of soybean roots in response to p-coumaric and p-hydroxybenzoic acids. Braz. Arch. Biol. Technol. 46, 193-198.

dos Santos, W.D., Ferrarese, M.L.L., Nakamura, C.V., Mourao, K.S.M., Mangolin, C.A., Ferrarese-Filho, O., 2008. Soybean (Glycine max) root lignification induced by ferulic acid. The possible mode of action. J. Chem. Ecol. 34,1230-1241.

dosSantos, W.D., Ferrarese, M.D.L., Finger, A., Teixeira, A.C.N., Ferrarese, O., 2004. Lignification and related enzymes in Glycine max root growth-inhibition by ferulic acid. J. Chem. Ecol. 30,1203-1212.

Einhellig, F.A., 1986. Mechanisms and modes of action of allelochemicals. In: Putnam, A.P., Teng, C.S. (Eds.), The Science of Allelopathy. John Wiley & Sons, New York pp. 170-188.

Einhellig, F.A., 1995. Mechanism of action of allelochemicals in allelopathy. In: Inderjit, Dakshini K.M.M., Einhellig, F.A. (Eds.), Allelopathy, Organisms, Processes and Applications. ACS Symposium Series, vol. 520. American Chemical Society, Washington, DC, pp. 96-116.

Einhellig, F.A., 2002. The physiology of allelochemicals action: clues and views. In: Reigosa, M.J., Pedrol, N. (Eds.), Allelopathy from Molecules to Ecosystems. Science Publisher Inc., Enfield, NH, pp. 1 -23.

El-Rokeik, K.G., 2007. Evaluating the physiological influence of benzoic and cinnamic acids, alone or in combination on wheat and some infested weeds comparing with the herbicide isoproturon. Ann. Agric. Sci. 52, 45-58.

El-Rokiek, K.G., Eid, R.A., 2009. Allelopathic effects of Eucalyptus citriodora on amaryllis and associated grassy weed. Planta Daninha 27, 887-899.

Galindo, J.C.G., Hernandez, A., Dayan, F.E., Tellez, M.R., Macias, FA, Paul, R.N., Duke, S.O., 1999. Dehydrozaluzanin C, a natural sesquiterpenolide, causes rapid plasma membrane leakage. Phytochemistry 52, 805-813.

García-Sánchez, M., Garrido, I., de Jesús Casimiro, I., Casero, P.J., Espinosa, F., García-Romera, I., Aranda, E., 2012. Defence response of tomato seedlings to oxidative stress induced by phenolic compounds from dry olive mill residue. Chemosphere 89, 708-716.

GmerekJ., Politycka, B., 2011. Response of maize, pea and radish roots to allelochemicals stress. Acta Biol. Cracov. Ser. Bot. 53, 32-37.

Goel, A., Sheoran, L.S., 2003. Lipid peroxidation and peroxide scavenging enzymes in cotton seeds under natural ageing. Biol. Plant. 46, 429-434.

Hachinohe, M., Matsumoto, H., 2005. Involvement of reactive oxygen species generated from melanin synthesis pathway in phytotoxicity of L-DOPA. J. Chem. Ecol. 31, 237-246.

Hare, P.D., Cress, W.A., van Staden, J., 1998. Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ. 21, 535-553.

Herrig, V., DeLourdes, M., Ferrarese, M.D.L., Suzuki, L.S., Rodrigues, J.D., Ferrarese, O., 2002. Peroxidase and phenylalanine ammonia-lyase activities, phenolic acid contents and allelochemicals inhibited root growth of soybean. Biol. Res. 35,59-66.

Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. 347,1-32 (Berkley).

Hoagland, R.E., Zablotowicz, R.M., Reddy, K.N., 1996. Studies of the phytotoxicity of sapo-nins on weed and crop plants. In: Waller, G.R., Yamaski, K. (Eds.), Saponins Used in Food and Agriculture. Plenum Press, New York, USA, pp. 57-73.

Jefferson, L.V., Pennacchio, M., 2003. Allelopathic effects of foliage extracts from four Chenopodiaceae species on seed germination. J. Arid Environ. 55,275-285.

Kaur, S., Singh, H.P., Mittal, S., Batish, D.R., Kohli, R.K., 2010. Phytotoxic effects of volatile oil from Artemisia scoparia against weeds and its possible use as a bioherbicide. Ind. Crop Prod. 32, 54-61.

Kazuyuki, W., Takayuki, H., Seiichiro, K., 1997. Abscisic acid suppresses the increases in cell wall-bound ferulic and diferulic acid levels in dark-grown wheat (Triticum aestivum L.) coleoptiles. Plant Cell Physiol. 38,811-817.

Kirby, M.F., Sheahan, D.A., 1994. Effects of atrazine, isoproturon and mecoprop on the macrophyte Lemna minor and the alga Scenedesmus subspicatus. Bull. Environ. Contam. Toxicol. 53,120-126.

Krystyna, O., Bailly, C., Gniazdowska, A., Come, D., Corbineau, F., Bogatek R., 2007. Induction of oxidative stress by sunflower phytotoxins in germinating mustard seeds. J. Chem. Ecol. 33,251-264.

Lara-Núñez, A., Sánchez-Nieto, S., Luisa Anaya, A., Cruz-Ortega, R., 2009. Phytotoxic effects of Sicyos deppei (Cucurbitaceae) in germinating tomato seeds. Physiol. Plant. 136, 180-192.

Latkowska, E., Bialczyk, J., Lechowski, Z., Czaja-Prokop, U., 2008. Responses in tomato roots to stress caused by exposure to (+)-usnic acid. Allelopathy J. 21, 239-252.

Lichtenthaler, H., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591-592.

Lutts, S., Kinet, J.M., Bouharmont, J., 1996. NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann. Bot 78, 389-398.

Mac Donald, M.J., D'Cunha, G.B., 2007. A modern view of phenylalanine ammonia-lyase. Biochem. Cell Biol. 85, 273-282.

Macias, FA, Molinillo, J.M.G., Galindo, J.C.G., Varela, R.M., Simonet, A.M., Castellano, D., 2001. The use of allelopathic studies in the search for natural herbicides. J. Crop. Prod. 4, 237-255.

Mishra, S., Srivastava, S., Tripathi, R.D., Govindrajan, R., Kuriakose, S.V., Prasad, M.N.V., 2006. Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L. Plant Physiol. Biochem. 44, 25-37.

Munne-Bosch, S., Alegere, L., 2004. Die and let live: leaf senescence contributes to plant survival under drought stress. Funct Plant Biol. 31, 203-216.

Musser, DA, Oseroff, A.R., 1994. The use of tetrazolium salts to determine sites of damage to the mitochondrial electron transport chain in intact cells following in vitro photo-dynamic therapy with Photofrin II. Photochem. Photobiol. 59, 621 -626.

Mustafa, M.G., 1990. Biochemical basis of ozone toxicity. Free Radic. Biol. Med. 9, 245-265.

Nazif, U., Ul Haq, I., Safdar, N., Mirza, B., 2013. Physiological and biochemical mechanisms of allelopathy mediated by the allelochemical extracts of Phytolacca latbenia (Moq.) H. Walter. Toxicol. Ind. Health 1-7.

Neish, A.C., 1961. Formation of M- and P-coumaric acids by enzymatic deamination of the corresponding isomers of tyrosine. Phytochemistry 1, 1 -24.

Nugroho, L.H., Verberne, M.C., Verpoorte, R., 2001. Salicylic acid produced by isochorismate synthase and isochorismate pyruvate lyase in various parts of constitutive salicylic acid producing tobacco plants. Plant Sci. 161, 911-915.

Omezzine, F., Haouala, R., 2013. Effect of Trigonella foenum-graecum L. development stages on some phytochemicals content and allelopathic potential. Sci. Hortic 160,335-344.

Omezzine, F., Ladhari, A., Nefzi, F., Harrath, R., Aouni, M., Haouala, R., 2012. Induction and flow cytométrie identification of mixoploidy through colchicine treatment of Trigonella foenum-graecum L. Afr. J. Biotechnol. 98,16434-16442.

Omezzine, F., Bouaziz, M., Simmonds, M.S.J., Haouala, R., 2014. Variation in chemical composition and allelopathic potential of mixoploid Trigonella foenum-graecum L. with developmental stages. Food Chem. 148,188-195.

Ozeki, Y., Komamine, A., 1985. Effects of inoculum density, zeatin, and sucrose on antho-cyanin accumulation in a carrot suspension culture. Plant Cell Tissue Organ Cult. 5, 45-53.

Patterson, D.T., 1981. Effects of allelopathic chemicals on growth and physiological responses of soybean (Glycine max). Weed Sci. 29, 53-59.

Pedrol, N., González, L., Reigosa, M.J., 2006. Allelopathy and abiotic stress. In: Reigosa, M.J., Pedrol, N., González, L. (Eds.), Allelopathy: A Physiological Process with Ecological Implications. Springer, The Netherlands, pp. 171-209.

Politycka, B., 1998. Phenolics and the activities of phenylalanine ammonia-lyase, phenol-B-glucosyltransferase and B-glucosidase in cucumber roots as affected by phenolic allelochemicals. Acta Physiol. Plant. 20,405-410.

Poppe, L., Rétey, J., 2005. Friedel-Crafts-type mechanism for the enzymatic elimination of ammonia from histidine and phenylalanine. Angew. Chem. Int. Ed. Engl. 44, 3668-3688.

Quettier, D.C., Gressier, B., Vasseur, J., Dine, T., Brunet, C., Luyckx, M.C., Cayin, J.C., Bailleul, F., Trotin, F., 2000. Phenolic compounds and antioxidant activates of buckwheat (Fagopyrum esculentum Moench) hulls and flour. J. Ethnopharmacol. 72, 35-42.

Rashid, M.H., Takashi, A., Uddin, M.N., 2010. The allelopathic potential of Kudzu (Pueraria montana). Weed Sci. 58,47-55.

Reid, P.D., Marsh, J.R., 1969. Gibberellic acid promoted activity of L-phenylalanine ammonia-lyase activity in several plant species. Z. Pflanzenphysiol. 61,170-172.

Reid, P.D., Evelyn, A.H., Herbert, V.M., 1972. L-Phenylalanine ammonia-lyase (Maize). Plant Physiol. 50, 480-484.

Rice, E.L., 1984. Allelopathy, Second edition. Academic Press, Inc., Orlando pp. 1-7.

Sakthivel, N., Amududha, R., Muthukrishnan, S., 2002. Production of phytotoxic metabolites by Sarocladium oryzae. Mycologology Res. 106,609-614.

Sampietro, DA, Marta, A.V., Maria, I.I., 2006. Plant growth inhibitors isolated from sugarcane (Saccharum officinarum) straw. J. Plant Physiol. 163, 837-846.

Sánchez-Moreiras, A.M., Pedrol, N., González, L., Reigosa, M.J., 2008. 2-(3H)-benzoxazolinone (BOA) induces loss of salt tolerance in salt-adapted plants. Plant Biol. 11, 582-590.

Schopfer, P., Plachy, C., Frahry, G., 2001. Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide & hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid. Plant Physiol. 125, 1591-1602.

Shann, J.R., Blum, U., 1987. The utilization of exogenously supplied ferulic acid in lignin biosynthesis. Phytochemistry 26,2977-2981.

Siddiqui, Z.S., 2007. Allelopathic effects of black pepper leachings on Vigna mungo (L.) Hepper. Acta Physiol. Plant. 29, 303-308.

Singh, H.P., Batish, D.R., Kaur, S., Ramezani, H., Kohli, RK, 2002. Comparative phytotoxicity of four monoterpenes against Cassia occidentalis. Ann. Appl. Biol. 141,111-116.

Singh, H.P., Batish, D.R., Setia, N., Kohli, R.K., 2005. Herbicidal activity of volatile essential oils from Eucalyptus citriodora against Parthenium hysterophorus. Ann. Appl. Biol. 146, 89-94.

Singh, H.P., Batish, D.R., Kaur, S., Arora, K., Kohli, R.K., 2006. Alfa pinene inhibits growth and induces oxidative stress in roots. Ann. Bot. 98,1261-1269.

Singh, A., Singh, D., Singh, N., 2009. Allelochemical stress produced by aqueous leachate of Nicotiana plumbaginifolia Viv. Plant Growth Regul. 58,163-171.

Singh, N.B., Sunaina, K. Yadav, Amist, N., 2013. Phytotoxic effects of cinnamic acid on cabbage (Brassica oleracea var. capitata). J. Stress Physiol. Biochem. 9, 307-317.

Stowe, R.P., Koenig, D.W., Mishra, S.K., Pierson, D.L., 1995. Non destructive and continuous spectrophotometry measurement of cell respiration using a tetrazolium-formazan microemulsion. J. Microbiol. Methods 22,283-292.

Stumpf, D.K., 1984. Quantification and purification of quaternary ammonium compounds from halophyte tissue. Plant Physiol. 75, 273-274.

Takayoshi, H., Kawamura, A., 1964. Enzymes of aromatic biosynthesis. Modem Methods of Plant Analysis, 7. Springer Verlag, Berlin, Gortingen & Heidelberg, pp. 260-285.

Tanatson, P., Udomporn, P., Umporn, S., Montinee, T., Patchanee, C., Chamroon, L., 2013. Phytotoxic effects of essential oil from Cymbopogon citratus and its physiological mechanisms on barnyardgrass (Echinochloa crus-galli). Ind. Crop. Prod. 41,403-407.

Teresa, M., Esperanza, M., Maria, A.M.-C., Francisco, J.L.-A., 1998. Effects of gibberellic acid (GA3) on strawberry PAL (phenylalanine ammonia-lyase) and TAL (tyrosine ammonia-lyase) enzyme activities. J. Sci. Food Agric. 77, 230-234.

Thapar, R., Singh, N.B., 2006. Effects of leaf — residues of Croton bonplandianum on growth and metabolism of Parthenium hysterophorus. Allelopathy J. 18, 255-266.

Velioglu, Y.S., Mazza, G., Gao, L., Oomah, B.D., 1998. Antioxidant activity and total pheno-lics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46, 4113-4117.

Verdisson, S., Couderchet, M., Vernet, G., 2001. Effects of procymidone, fludioxonil and pyrimethanil on two non-target aquatic plants. Chemosphere 44,467-474.

Wildermurth, M.C., 2006. Variations on a theme: synthesis and modification of plant benzoic acids. Curr. Opin. Plant Biol. 9,288-296.

Yang, C.M., Chang, I.F., Lin, S.J., Chou, C.H., 2004. Effects of three allelopathic phenolics on chlorophyll accumulation of rice (Oryza sativa) seedlings: II. Stimulation of consumption orientation. Bot Bull. Acad. Sin. 45,119-125.

Yang, C.M., Lee, C.N., Chou, C.H., 2002. Allelopathic phenolics and chlorophyll accumulation. Effects of three allelopathic phenolics on chlorophyll accumulation of rice (Oryza sativa) seedlings: I. Inhibition of supply-orientation. Bot. Bull. Acad. Sin. 43, 299-304.

Zeng, L., Shannon, M.C., Lesch, S.M., 2001. Timing of salinity stress affects rice growth and yield components. Agric. Water Manag. 48,191-206.

Zou, L.Y., Ogweno, J.O., Sun, Y., Shi, K., Zhou, Y.H., Yu, J.Q., 2006. Autotoxic potential of root exudates and associated phenolics in watermelon. Allelopathy J. 18,103-109.