1. Background
Contamination of soil with As and As-containing salts is a global environmental problem. As-based pesticides, fertilizers, metal processing industries and coal combustion units are some of the main sources of As pollution (Meharg and Whitaker, 2002; Liao et al., 2004). Pollution of groundwater with As is increasing at an unprecedented rate in some South Asian countries, especially Bangladesh and India (Ghosh et al., 2006). Irrigation of agricultural fields with As polluted water has significantly increased As levels in soil (Marin et al., 1992). Consumption of food grown in As-contaminated soil causes many health problems such as cancer, cardio-vascular disease and neurological disorders (Gadepalle et al., 2008). As polluted water has the potential to cause severe skin allergies, dermatological lesions and other health related problems.
 
Persistence of As in the environment for long duration is resulting in decrease in yield and quality of many important agricultural crops (Rashid et al., 2004). As is taken up from the soil by plant roots and is transferred to higher trophic levels via food chain (Zhang et al., 2002). Experiments carried out on a variety of crop species have demonstrated that As contamination in soil causes adverse effects on plant biomass productivity and yield (Carbonell-Barrachina et al., 1997). As uptake by plants and its effect on plant nutrition has been investigated in detail for various species such as Brassica juncea, Oryza sativa, Pteris vittata and Spartina alterniflora (Carbonell et al., 1998; Abedin and Meharg, 2002; Chaturvedi, 2006; Fayiga et al., 2007).

EO yielding plants are important cash crops and are sources of bioactive constituents that have medicinal value. These plants are considered to be hardy and grow safely on metal-polluted soils around smelters and soil contaminated with heavy metals (Zheljakov and Nielsen, 1996; Salamon, 2008). Studies conducted on some important medicinal plants such as Bidens tripartite, Leonurus cordiaca, Marrubium vulgare, Melissa officinalis and Origanum heracleoticum clearly depict that no severe phytotoxic symptoms were observed in morphology, EO percentage and yield of these plants (Zheljakov et al., 2008). Similarly, EO yield of Mentha piperita and Ocimum basilicum is also not affected by the treatments of Cd, Pb and Cu in soil. However, application of these heavy metals can alter the composition of EO (Zheljakov et al., 2006).

Ocimum basilicum L. (Lamiaceae), commonly known as sweet basil is an annual aromatic medicinal herb native to India and other regions of Asia. In India, basil is mainly cultivated in Assam, Bihar, Uttar Pradesh and West Bengal where soil is severely polluted with As (Heikens, 2006; Rao et al., 2007). It grows luxuriantly in variety of soil types and agroclimatic conditions (Begum et al., 2002). EO derived from basil is widely utilized in high-grade perfumes, aromatherapy, flavoring liquors and as herbal spice (Bahl et al., 2000; Kumar et al., 2004). EO contains biologically active constituents that possess antimicrobial (Elgayyar et al., 2001), fungistatic (Reuveni et al., 1984), insecticidal (Bowers and Nishida, 1980) and allelopathic properties (Rice, 1979). Traditionally, basil has been used for the treatment of headache, cough, diarrhoea, constipation, warts and kidney malfunction (Politeo et al., 2007).  The yield and composition of EO is dependent on many environmental factors, agrochemical practices and the type of cultivar (Jirovetz et al., 2003).

Leaves of O. basilicum bear non-glandular and glandular trichomes on both the abaxial and adaxial surfaces. Non-glandular trichomes are uniseriate, pointed, straight or hook-like and their function is to confer defence to plants. Glandular trichomes are responsible for biosynthesis, secretion and accumulation of EO (Fischer et al., 2011). According to Wolff et al. (2012) high concentration of heavy metal in soil affects the structural integrity of the trichomes. However, there is no detailed experimental evidence to address how trichomes respond to high As levels in soil, and how the alteration of trichome structure influences the EO yield and quality. Studies on how ultrastructure of glandular trichomes is affected in treated plants and how cell organelles of functional importance such as endoplasmic reticulum (ER), mitochondria and plastids respond to high As concentrations has not been carried out in relation to As toxicity.

The present study was designed to investigate: (i) effect of different As concentrations in soil on EO yield and composition of basil plants; (ii) impact of As in the soil on morphology and ultrastructure of EO secreting glandular trichomes. The study has been undertaken to understand the EO secretion and glandular trichome function in uncontaminated (control) and As- contaminated soil.

2. Results
1.1.  Effect of As on growth
Table 1 shows that O. basilicum plants exposed to increasing As concentrations showed progressive decrease in the vegetative growth i.e. shoot length and shoot biomass in terms fresh and dry weight. Highest reduction was observed at 150 mg/kg As. Shoot length was significantly reduced by 34.65%, compared to control, while difference between other treatments was not significant. Shoot fresh and dry mass accumulation was inhibited by 31.78 and 27% respectively at 150 mg/kg As. However, an increase in shoot length, fresh and dry weight was observed at 10 mg/kg As.
 

1.2. Effect of As on EO yield and composition
Hydrodistillation of leaf extract from the three month old basil plants was carried out. EO yield of the control plants was 0.20% (w/w). At 10 and 50 mg/kg As, EO yield significantly increased 3.5-4 times to reach 0.77 and 0.8% respectively, whereas at 150 mg/kg As the yield significantly reduced up to 0.08% (Figure. 1). EO components estimated in control plants were linalool (0.0013 mg/g DW), methyl cinnamate (0.013 mg/g DW), camphor (0.0072 mg/g DW), 1,8-cineol (0.009 mg/g DW) and methyl eugenol (0.028 mg/g DW). Linalool concentration increased 3-4 times under 10-150 mg/kg As as compared to the control. Methyl cinnamate concentration at 10 mg/kg As showed down regulation up to 72.30%, whereas at 50 and 150 mg/kg As it was not detected. Concentration of 1,8-cineol and methyl eugenol decreased by 67.7 and 96% respectively at 150 mg/kg As in comparison  to EO extracted from leaves of control plants. Camphor was severely affected by As toxicity and not detected at all in any of the As-treated plants (Table 2).
 

1.3. Effect of As on trichome  density and development
1.3.1.  Trichome density
 On the adaxial surface, trichome density increased 2-3
times at 10 and 50 mg/kg As in comparison to control plants. At 150 mg/kg As, trichome density decreased by 34%. Trichome density on abaxial epidermis increased at 10 mg/kg As by 66%, while at 50 and 150 mg/kg As it decreased by 67 and 34 % respectively as compared to control (Figure. 2).
 

1.3.2.  Trichome structure and ultrastructure
LM studies showed that glandular trichomes on the leaves of control plants were globular, with a stalk of 1-3 cells and a head of four secretory cells (Figure. 3A). At 10 and 50 mg/kg As in soil structural collapse of the trichome head by disorganization of the secretory cells and folding of their cell walls was observed. Trichomes of plants treated with 150 mg/kg As showed senescence-like symptoms. Head cells showed mushroom-like appearance, partially protruding out of the epidermal depression (Figure. 3D). SEM investigations revealed an equatorial line of weakness around the head of the trichome in leaves of control plants (Figure. 4A). The cuticle ruptured along this line, and the subsequent collapse of the subcuticular cavity led to the release of exudates. Early maturity of trichomes was observed in As treated plants. Disintegration of the secretory cells and folding of the cell walls was observed in trichomes at 10, 50 and 150 mg/kg As treatments. Exposed head cells with no cuticle were noticed at 50 mg/kg As (Figure. 4C). A large number of deeply grooved trichomes was common at 150 mg/kg As (4D), a typical behaviour of mature trichomes (Werker, 1993). Wax deposition was more clearly visible on leaf surface (Figure. 4D). Distortion of epidermal cells was observed with increased concentration of As. Non-glandular trichomes also lost the structural integrity with higher doses of As in soil.
 

TEM studies provided further insights into the secretory cells of trichomes. Well-developed central nucleus and dense cytoplasm was observed in the head cells of control plants. Large vacuoles and mitochondria having well developed cristae were seen all over (Figure. 4E). Rough endoplasmic reticulum (RER) and dictyosomes were also distinctly visible, signifying that cells were actively involved in EO secretion. At 10 mg/kg As no significant variation was observed in head cells in comparison to control. Well developed nucleus with electron dense chromatin material was observed in the cells (Figure.4F). However, at 50 mg/kg As, plasmodesmatal connections were more prominent on the secretory cell wall and large mitochondria were seen (Figure 4. 4G). At 150 mg/kg As, glandular trichomes showed various changes at structural and ultrastructural level. The sectretory cells showed less number of vacuoles. RER were well represented but size of mitochondria was observed to be reduced (Figure. 4H).
 

 
2. Discussion
Results of the present study clearly demonstrate that the growth of O. basilicum significantly decreased as a result of higher As phytotoxicity, though at lower As concentrations the effect was not significant. An increase in plant growth at 10 mg/kg As in soil was observed. Results are in agreement with the study on Scutellaria biacalensis, an important herbal plant used in traditional Chinese medicine where low levels of As in soil stimulated the growth and development (Cao et al., 2009). This positive response at lower As concentrations can be linked to phosphorous uptake. Phosphate and arsenate are taken into plant roots by a common carrier. However, the phosphate/plasma membrane carrier has much higher affinity for phosphate than arsenate (Meharg and Macnair, 1990). Phosphate is also reported to be an efficient competitive inhibitor of arsenate uptake (Meharg and Macnair, 1990). At low soil As concentration, displacement of soil phosphate by arsenate increases the availability of phosphate to the plants, which results in the increase of growth.
 
Four-fold increase in EO yield in plant tissues was observed when As concentration in the soil was raised up to 50 mg/kg, whereas at 150 mg/kg As the oil yield significantly decreased by about 60% in comparison to control. Heavy metal-induced enhancement in EO yield at low concentration in growth medium has been reported earlier in other EO yielding species such as Matrica chamomilla (Nasiri et al., 2010), Mentha piperita (Prasad et al., 2010) and Salvia officinalis (Stancheva et al., 2009). Increase in the EO content by the application of heavy metals is not properly understood, but the change has been attributed to the effect of metallic elements on the enzyme activity and carbon metabolism which further affects the EO synthesis pathways (Prakash and Kardage, 1980). Increase in EO production and density of EO releasing trichomes in O. basilicum has been observed in the present study at moderate As levels. This can be a partial explanation for the observed higher oil content per unit leaf dry weight. According to Charles et al. (1990) EO accumulation increases indirectly by interfering with net assimilation rate of nutrients in plants or by unequal partitioning and distribution of resources for growth and differentiation. EO production in basil leaves was strongly inhibited at 150 mg/kg As. Reduction in photosynthesis and/or additional changes in metabolic system are probably responsible for this inhibition. According to Croteau and Johnson (1984) EO biosynthesis takes place in epidermal oil glands that are carbon heterotrophic and thus depend on the adjoining photosynthesizing cell for a continuous supply of carbon precursors. EO production is also dependent on availability of various nutrient ions in soil. Any disruption in the nutrient balance reduces the oil production as observed in basil plants exposed to higher level of As in soil. Such results have also been observed in plants growing in saline soils.  Reduction in EO yield due to high dose of 100 mM NaCl has been observed in Salvia officinalis (Ben Taarit et al., 2009) and Origanum majorana (Baatour et al., 2010). As in soil disturbs the ionic balance and availability of nutrient ions to plant systems as observed in response to salinity. Comparison of plant biomass and oil content at different treatments revealed that As stress showed more severe effect on biomass than on oil content. This low reduction of oil content is certainly an advantage for EO plants. Biswas et al. (2011) in a detailed review have attributed the changes in oil yield in various EO plants to cascading effects of stress imposed on account on salt, heavy metal or water availability. Stress encountered by the plants affects the presence and availability of nutrient elements, and hence the secretory pathway.

Ultrastructual studies of secretory structures of trichomes showed early maturity in plants grown in As amended soil.  At 150 mg/kg As, structure of head cell changed from globular to mushroom-like and the trichomes were seen embedded into the epidermal surface. Such changes are generally observed in trichomes at post secretory stage (Gravano et al., 1998). Werker et al. (1993) also observed that in O. basilicum glandular trichomes were embedded in the epidermis when trichomes attain maturity and proceed to senescence. Premature senescence of trichomes is distinctly observed in the present studies in response to As toxicity.

At 10 mg/kg As trichome ultrastructure did not show any variation in comparison to trichomes of control plants. At both the treatments, the head cells showed highly-organized cytoplasm, large nuclei, and many mitochondria. These features are typical of secretory tissues with high metabolic activity. Secretion is an active process in which energy is used for metabolic compartmentation, ion extrusion, or biosynthesis of products (Fahn, 1988). Hence, presence of a large number of mitochondria in glandular trichomes is indicative of active secretion.  Presence of large vacuoles is related to the storage of metabolites and ions in the secretory apparatus (Figueiredo and Pais, 1994). At 50 mg/kg As, trichomes of O. basilicum showed extensive RER and large mitochondria, indicating that the activity was higher in glandular cells that stimulated the production of EO. The existence of more prominent plasmodesmata connecting the cytoplasm of the secretory cells indicates enhanced intercellular transport of compounds within the trichome.  Mitochondrial aberration in the form of inflated cristae and less electron dense cytoplasm was seen at 150 mg/kg As. Underdeveloped organelles in the trichomes at 150 mg/kg As could be the possible reason for low yield in EO. This clearly demonstrates that higher doses of As affect the cells and disrupt their metabolic machinery. The variation observed in yield are manifestations of changes observed at cellular and subcellular levels.

Characteristic basil aroma appears due to 1,8-cineol, methyl cinnamate and linalool (Lee et al., 2005). Camphor, 1,8-cineol and linalool are also known to be biologically active components (Morris et al., 1979). These compounds possess antimicrobial and antioxidative properties with high therapeutic value (Raseetha et al., 2009). GC analysis revealed that linalool, the major constituent in O. basilicum was not affected by exposure to As, but relative concentration of methyl eugenol, methyl cinnamate, 1,8-cineol and camphor diminished when plants were subjected to As stress (Table 2). Such a change in constituents of EO can be considered detrimental to oil quality. Low concentration of 1,8-cineol and absence of camphor at higher As levels depict that there is a compromise in defense potential as these two constituents are involved in allelopathic reactions (Rice, 1979). Compounds responsible for typical aroma of basil are affected due to As toxicity, which also degrades the antioxidative property of basil. Such changes in the EO composition in response to heavy metal stress have also been reported in Rosmarinus officinalis (Deef, 2007), Mentha arvensis (Prasad et al., 2010) and Salvia officinalis (Stancheva et al., 2009). Compromise on quality of therapeutically important compounds reflect inactivation of enzymes or redirection of metabolic functions to maintain growth (Murch et al., 2003).
 
3. Material and Methods
3.1. Plant material, growth conditions and treatments
Seeds of Ocimum basilicum were procured from Germplasm Conservation Division, National Bureau of Plant Genetic Resources (NBPGR), New Delhi (India). Pot culture experiments were conducted in Botanical Garden, University of Delhi, Delhi (India).  Seeds were sown in October in pots (38 cm diameter) filled with 4 kg air dried soil and compost in equal ratio. Soil was clay loam, pH 7.2, NO3 N 125 mg/kg, available P 0.5 mg/kg and K 120 mg/kg. Air-dried soil was amended with disodium hydrogen arsenate [Na2HAsO4.7H2O] at concentrations of 10, 50 and 150 As mg/kg soil, each with three replicas. Pots without As served as control. Twenty seeds were sown in each pot and five uniformly growing seedlings per pot were retained for further studies. Plants were grown under natural conditions of light, temperature and humidity.
 
Plants were harvested after four months of growth period. Whole plants were scooped out. Fresh weight of the shoot system was taken. Plant tissue was oven dried at 72 o C and weight was recorded.
 
3.2. EO extraction
Fifty grams of dried shoots (leaves and stems) were subjected to steam distillation-extraction for 4 h according to the protocol given by Rao et al. (2005) with slight modifications. The condensate obtained from distillation was collected and divided into sub-samples. Each sub-sample was mixed with 100 ml hexane to trap the dissolved EO. EO obtained from the samples was dried over anhydrous sodium sulphate to make it moisture free. Quantity of EO was measured by calibrated burette. Percentage EO yield of tissue samples was calculated by the formulae given by Rao et al. (2005):
 
EO yield (%) = Amount of EO recovered (g) / Amount of crop biomass distilled (g) x 100

3.3. EO analysis
EO analysis was carried out by gas chromatography using Shimadzu GC 2014 equipped with flame ionization detector (FID) and an electronic pressure control (EPC) injector. Helium was used as the carrier gas with a flow rate 1.2 ml/min through Restek DB-5 capillary column (30 m long x 0.53 µm diameter and film thickness 0.5 µm). One µl oil sample was injected, diluted in hexane (0.05 ml of oil into 0.95 ml of GC standard grade hexane) and the samples were analyzed by GC.  Analysis was programmed for 15 min at 60oC, rising up to 240oC at the rate of 5ºC/min. EO components were identified by comparing their retention times with those of authentic standards  of linalool, 1,8- cineol, methyl cinnamte, methyl eugenol and camphor (CDH and Merck, India) run under the same conditions (Kovats, 1965). Concentration of each component in oil sample was calculated using the following equation (Lee et al., 2005).

Concentration (mg/g) = Weight of extract (without solvent) x GC peak area % 100 (mg) / Weight of plant material (g)

3.4.  Trichome study
Micromorphological observations were carried out on fresh basil leaves by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TEM was used to reveal ultrastructural details of trichomes.

3.4.1. Light microscopy (LM): Hand-peel mounts were prepared to study trichome density per unit leaf area on both adaxial and abaxial surfaces. For paraffin wax sectioning, plant material was fixed in FAA (formaldehyde: glacial acetic acid: ethanol, 1:1:18, v/v/v) for 24 h. For dehydration the fixed plant parts were passed through tertiary butyl alcohol (TBA) series. Embedding was done in paraffin wax following the procedure of O’Brien and McCully (1981). Wax blocks containing embedded material were trimmed and mounted on wooden blocks for sectioning with the help of Riechert rotary microtome. Sections of 8 µm thickness were cut, dewaxed and dehydrated through ethyl alcohol-xylene series and stained with 1% safranin and 1% astra blue combination. Mounting was done in DPX and observed under Nikon photomicroscope.

3.4.2. Scanning electron microscopy (SEM): Leaf segments were fixed in Karnovsky’s fixative, buffered with 0.1 M sodium phosphate buffer at pH 7.4 for 6-8 h at 4oC. After washing in the buffer the material was dehydrated in a graded ethanol series, critical point dried with CO2, mounted in stubs and coated with a thin layer of gold (Serrato-Valenti et al., 1997). Sections were observed under LEO 435 VP scanning electron microscope at 15 KV.

3.4.3. Transmission electron microscopy (TEM): For ultrastructural investigation, leaves were trimmed and small pieces from margin up to midrib were fixed in Karnovsky’s fluid, buffered with 0.1 M sodium phosphate buffer at pH 7.4 for 6-8 h at 4 oC and post fixed in 1% osmium tetraoxide. After dehydration in ethanol series, the material was embedded in Epon-Araldite resin. Sections were conventionally stained with uranyl and lead citrate (Ascensão et al., 1997) and examined under Philips 200 transmission electron microscope at 80 KV.

3.5. Statistical analysis
Data were subjected to statistical analysis using software package SPSS 10.0 (Statistical Package for Social Sciences). One-way analysis of variance (ANOVA) followed by multiple comparison least significance difference (LSD) was employed to check the significance of the differences between the treatments at p ≤ 0.05.

Conclusions
As in growth medium decreased both growth and biomass of aerial parts in O. basilicum. EO yield showed an up regulation at 10 and 50 mg/kg As, but it decreased at 150 mg/kg As.  Linalool, the main component was augmented in all As treatments. Methyl cinnamate and camphor diminished under higher As concentrations. A strong positive correlation between trichome density and EO yield was noticed. Alteration in the structure and ultrastructure of glandular trichomes demonstrated that trichomes under As stress achieved precocious senescence, which further led to malfunctioning of secretory machinery. However, there is further need to carry out experiments in natural and controlled conditions to understand the exact mechanism by which the metabolic pathways are altered. Data provides an early indication that environmental pollutants like As affect the medicinal quality of O. basilcum. The development of optimized agricultural practices is essential for the sustainable cultivation of O. basilicum and adequate yield of EO.
 
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