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Journal of Stress Physiology & Biochemistry, Vol. 7 No. 4 2011, pp. 113-121 ISSN 1997-0838 Original Text Copyright © 2011 by Kharadi, Upadhyaya, Upadhyay and Nayak
ORIGINAL ARTICLE
Differential responses of plumbagin content in Plumbago zeylanica L. (Chitrak) under controlled water stress treatments
Kharadi R., Upadhyaya S.D., Upadhyay A. and Nayak Preeti Sagar*
Department of Plant Physiology, College of Agriculture, Jawaharlal Nehru Krishi Vishwavidalaya (JNKVV), Jabalpur 482 004 (M.P.) India
*E-mail: preetisagarnayak@rediffmail.com
Received August 30, 2011
A pot experiment was conducted on Plumbago zeylanica L. (Chitrak) under controlled water stress environment in greenhouse during the kharif season. The experiment was laid out in completely randomized design with five treatments of different water stress levels i.e. control, 20%, 40%, 60% and 80% and four replications. Out of five stress levels, 80% water stress has influenced root length, dry herbage, plumbagin, potassium and proline content. In control conditions the plant height, number of leaf, total leaf area, stomatal conductance, transpiration rate, photosynthesis, CO2 utilization, H2O utilization and chlorophyll were found to be maximum. The impact of water stress on plumbagin content has shown increase trend with respect to different water stress levels that is maximum at 80 % and minimum at control.
Key words: Plumbago zeylanica, water stress, plumbagin
ORIGINAL ARTICLE
Differential responses of plumbagin content in Plumbago zeylanica L. (Chitrak) under controlled water stress treatments
Kharadi R., Upadhyaya S.D., Upadhyay A. and Nayak Preeti Sagar*
Department of Plant Physiology, College of Agriculture, Jawaharlal Nehru Krishi Vishwavidalaya (JNKVV), Jabalpur 482 004 (M.P.) India
*E-mail: preetisagarnayak@rediffmail.com
Received August 30, 2011
A pot experiment was conducted on Plumbago zeylanica L. (Chitrak) under controlled water stress environment in greenhouse during the kharif season. The experiment was laid out in completely randomized design with five treatments of different water stress levels i.e. control, 20%, 40%, 60% and 80% and four replications. Out of five stress levels, 80% water stress has influenced root length, dry herbage, plumbagin, potassium and proline content. In control conditions the plant height, number of leaf, total leaf area, stomatal conductance, transpiration rate, photosynthesis, CO2 utilization, H2O utilization and chlorophyll were found to be maximum. The impact of water stress on plumbagin content has shown increase trend with respect to different water stress levels that is maximum at 80 % and minimum at control.
Key words: Plumbago zeylanica, water stress, plumbagin
Plumbago zeylanica L. (Chitrak) is a semi climbing perennial herb that grows throughout India, Asia and Africa, also in Hawali, Virgin Island. It is found wild in peninsular India and in low elevations in Taiwan (Anonymous, 1962). It is a small perennial 0.5 to 1.5m tall, erect or climbing under shrub with flexible branches. Stems many, smooth, woody, striate, leaves simple, alternate glaborous, ovate, entire, oblong, 5 to 9.5 cm long and 2 to 6 cm broad. Flowers white, long, glandular, strike like racemes. Inflorescences stalk 20-30 cm
long branches on which tubular flowers are borne. Calyx is glandular and toothed while corolla tube 17-22cm long. Fruits cylindrical, small capsule, oblong enclosed in the calyx. Roots finger size thick, blackish purple bark and whitish internally with bitter smell. Flowering season is winter followed by fruiting after a month. The root contains a number of naphthaquinone derivatives (Plumbagin yellow, 3 Chloroplumbagin, 3, 3-biplumbagin, Elliptinone, Chitranone, Droserone, Zeylanone, Iso-zeylanon and Plumbazeylanone. Leaves and stem
contain volatile oil. The root possesses obortifacient and vesicant properties (Gupta, 2008). The whole plant and its roots have been used as folk medicine. Plumbagin is present in families of Plumbaginaceae and Droseraceae (Botanical Dermatology Database, 1991). Plumbagin has received an enormous amount of attention in pharmacological research, due to its antimalarial (Likhitwitayawuid et al., 1998), antimicrobial (Didry et al., 1998), anti-cancer, anti-carcinogenic, anti-mutagenic (Sugie et al., 1998) and chemotherapeutic activities (Hakura et al., 1994). It is used as diuretic, caustic and excellent of phlegmatic tumors and is useful in rheumatism. It is also used as an irritant of the skin, in the treatment of dyspepsia, piles, anasarca, diarrhea, skin diseases and in minute dosages in stimulant for liver (Gupta, 2008). Cultivation of medicinal plants under water stress conditions is an important factor for controlling levels of phytochemicals (Zheng et al.,
2007). Environmental stresses trigger a wide variety of plant responses, ranging from altered gene expression and cellular metabolism to changes in growth rates and crop yields. However, some studies on the use of water stress to enhance or increased the production of useful natural products in medicinal plants. Stress is measured in relation to plant survival, crop yield, growth (Bio mass accumulation) or the primary assimilation processes (CO2 and mineral uptake) which are related to overall growth. The alkaloid content in the plants is presumed to be the resultant of some abiotic stress.
The review literature reveals that the effects of moisture stress on plumbagin content in Plumbago zeylanica L. are lacking. Thus the present work was carried out to investigate the effects of water stress treatments on a number of growth parameters, biomass production and biochemical responses of the crop.
MATERIALS AND METHODS
Location and Duration: The pot experiment was conducted at the climate control Green house research area of Medicinal and Aromatic plants under the Department of Crop and Herbal Physiology, JNKVV, Jabalpur, (M.P) during the Rabi season 2007-2008. The soil of experimental pot was sandy loam. Date of transplanting was 15 th June 2007 and harvesting was carried out on 25th Feb 2008. Four plants of Plumbago zeylanica L. were transplanted in each pot applied with FYM, Biofertilizer and Inorganic fertilizer. Sampling was done at 15 day intervals for growth analysis, phenological and biochemical parameters. The sampling was done at 60, 75, 90,105, 120, 135 and 150 days after sowing.
Pot Experiment: The design was completely randomized design and experiment included five treatments of water regimes at field capacity (FC) with four replicates. The five water stress treatment are 20%, 40%, 60%, 80% and control.
• Control (FC).
• Mild Water Stress (20%).
• Moderate Water Stress (40%).
• Severe Water Stress (60%).
• Very Severe Water Stress (80%).
To calculate the field capacity, at the beginning of the experiments, pots were filled with known weight of mixture of sand: compost, saturated with water and allowed to drain freely for a period of 24 hours, until there was no change in weight. The difference between this weight and soil dry weight (DW) was used to calculate 100% of water holding capacity (WHC) (Liu and Stutzel, 2004). Before the beginning of water stress treatments, all pots watered to FC. At the beginning of the experiment, plants subjected to control water regime were
irrigated daily to maintain fully FC (well watered plants), while irrigation of the plants of 20%, 40%, 60% and 80% FC water regime treatments (water stressed plants) was withheld until the field capacity reached 20%, 40%, 60% and 80% FC respectively.
Growth Measurements: TPlant height (cm), number of leaf, total leaf area and LAI by Gardner et al, (1985), Biological yield /plant and root yield / plant. Photosynthesis etc were determined by using Infra Red Gas Analyzer of LI-COR Model LI-6400 portable photosynthesis system, USA.
Growth Parameters: Plant height (cm), number of leaf, total leaf area and LAI by Gardner et al, 1985. Photosynthesis etc were determined by using
Infra Red Gas Analyzer of LI-COR Model LI-6400 portable photosynthesis system, USA. Biological yield /plant, root yield / plant and
Biochemical parameters: Chlorophyll by
Yoshida et al., (1972), Potassium by Black (1965), Proline by Bates et al., (1973) and Plumbagin by HPTLC.
Statistical analysis: Analysis of observation taken on different variable was carried out to know the degree of variation among all the treatments. The pooled data was statistically analyzed through completely randomize design (Fisher, 1967).
plants under
Table 1 : Growth measurements and biomass production of Plumbago zeylanica L. water stress conditions in pot.
Treatments Plant No. of Total leaf Leaf Area Water Use
height Leaves area Index Effiency
Control 56.25 34.62 21.69 2.89 23.26
20% water stress 55.13 33.31 20.06 2.53 20.46
40% water stress 41.06 20.68 15.44 2.20 16.93
60% water stress 31.50 18.25 11.88 1.64 15.18
80% water stress 22.94 12.56 7.44 1.88 12.93
S.Em± 0.0071 0.0069 0.0084 0.0074 0.0083
C D 5% 0.0213 0.0209 0.0252 0.0222 0.0251
RESULTS AND DISCUSSION
The moisture stress treatment exhibited the significant influences over growth parameters. The maximum plant height was registered in control (56.2cm) and minimum was registered in 80% moisture stress (22.94 cm). The maximum number of leaves was recorded on control (34.62) and
minimum was registered in 80% moisture stress
(12.56) due to the turgor pressure of the cell. The maximum total leaf area was found in control (21.68) and minimum in 80% moisture stress (7.44). The maximum LAI was found in control (2.89) and minimum was registered in 80% moisture stress
(1.88). There is low increase in plant height under
extreme deficit possibly due to reduced cell turgor which affects cell division and expansion (Luvaha et al., 2008). The results of the study indicate that water deficit decreased leaf number, leaf area, leaf water content, shoot height, shoot dry weight and chlorophyll concentration. Decreases in leaf number and leaf area are common occurrences in water deficit stressed plants (Luvaha et al., 2008). Reduction in leaf number under extreme water deficit may have been due to reduction in leaf formation. Reduction in number of leaves can be a phenomenon by the plants to reduce transpiration surface hence water loss. Similar results have been observed in mango rootstock seedlings, which show
a decline in number of leaves due to drying or senescence of lower mature leaves (Luvaha et al.,
2008). Reduced leaf area decreases interception of solar radiation and consequently decreases biomass production for most crops (Masinde et al., 2005).
The maximum photosynthesis rate and stomatal conductance were found in control (10.66 mol m-2s-1
and 10.04 mol m-2s-1) and minimum in 80% water stress (3.87 mol m-2s-1 & 2.90 mol m-2s-1). Decreased number of stomata under higher moisture deficit condition has been reported by Xu and Zhou (2008) in grasses. Ghannoum e. al., (2003) and Ripley et al., (2007) suggested the reduction in leaf net photosynthetic assimilation by both stomatal and metabolic limitations under moisture stress situation.
plants under
Table 2 : Growth measurements and biomass production of Plumbago zeylanica L. water stress conditions in pot.
Treatments Net Photosynthesis Stomatal conductance CO2 Utilization Transpiration rate H2O Utilization
Control 10.66 10.04 9.55 0.46 1.14
20% water stress 9.86 9.50 9.28 0.41 1.07
40% water stress 6.81 6.93 6.89 0.38 0.74
60% water stress 4.39 5.15 5.10 0.31 0.69
80% water stress 3.87 2.90 3.94 0.33 0.57
S.Em± 0.0074 0.0077 0.0074 0.0071 0.0081
C D 5% 0.0224 0.0234 0.0222 0.0214 0.0245
The maximum CO2 and H2O utilization were found in control (9.55 ppm and 0.46 Kap) and minimum in 80% water stress (3.94 ppm and 0.33 Kap). The maximum transpiration rate and water use effiency were found in control (1.14 mol m-2s-1 and 23.26) and minimum in 80% water stress (0.57 mol m-2s-1 and 12.93).
Maximum root length was found in 80% water stress (16.3cm per plant) and minimum in control (5.4 cm per plant). The increased of root dry weight under drought conditions is in accordance with previous studies in other plant species (Sharp et al., 1990; Boutraa and Sanders, 2001; Liu and Stutzel, 2004). This might be explained by the functional balance theory, proposed by (Brouwer, 1963), that in plant imposed to limited water supply shoots will be checked sooner than that of roots because the latter are closer to the source of water supply limitation, leading to increase in root dry masses due to the increase of assimilates flow to belowground. Harris (1973) observed that retardation of shoot and
root growth by moisture stress.
Maximum dry herbage yield was found in control (17.64gm) followed by 20% water stress (17.45 gm) and minimum in 80% water stress (5.90 gm). Decrease in herbage yield under water stress has also been reported in other medicinal and aromatic plants (Singh- Sangwan et al., 2001; Fatima et al., 2002).
The maximum chlorophyll content was found in control (27.48%) followed by 20% water stress (23.79%) and minimum in 80% (5.54%).
Chlorophyll concentration reduced with increase in water deficit. This could be attributed to an increase in oxidative stress. Under more prolonged water deficit, dehydration of plant tissue can result in an increase in oxidative stress, which causes deterioration in chloroplast structure and an associated loss of chlorophyll. This leads to a decrease in the photosynthetic activity (Jafar et al., 2004). Reduction in chlorophyll concentration in water stressed plants could indirectly lead to a
decrease in photosynthetic activity. Total well water plants (Kirnak et al, 2001; Cengiz et al.,
chlorophyll content reduces by 55% compared to 2006).
Table 3: Biochemical profils of Plumbago zeylanica L. plants under water stress conditions in pot.
Treatments Chlorophyll (%) Herbage/ pod Root length (cm) Proline (%) Potassium (%) Plumbagin (%)
Control 27.48 17.64 5.40 1.70 1.40 0.0984
20% water stress 23.80 17.45 9.80 1.87 1.55 0.1034
40% water stress 18.47 15.65 13.40 2.35 1.68 0.1291
60% water stress 14.94 10.63 15.20 2.36 1.75 0.1323
80% water stress 5.54 5.90 16.30 2.39 1.89 0.2342
S.Em± 0.0070 0.0085 0.0416 0.0074 0.0085 0.0001
C D 5% 0.0211 0.0257 0.1255 0.0222 0.0257 0.0003
Figure 1: HPTLC chromatogram of plumbagin content in Plumbago zeylanica. (a) Plumbagin Standard (b) Plumbagin in sample.
The maximum proline content was in 80% water stress (2.39%) followed by 60% water stress (2.36%) and minimum in control (1.70%). Water stress induced the accumulation of free proline in plants may be part of a general adoption to water stress (Hare et al., 1998). Amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental stresses. In addition to its role as an osmolyte for osmotic adjustment, proline contributes to stabilizing sub-cellular structures (e.g., membranes and proteins), scavenging free radicals and buffering cellular redox potential under
stress conditions. It may also function as a protein compatible hydrotrope (Srinivas and Balasubramanian, 1995). The concentration of free proline of plumbagin stressed plants was significant increase in response to water stress compared to the control plants. Current study showed that the effect of water stress in pots experiments lead to significant increase in the total free proline. The obtained data were in agreement with (Wu et al., 2006) who stated that the content of free proline significantly increased under water stress of Rosmarinus officinalis L. Blum and Ebercon (1976) indicated that proline is regarded as a source of
energy, carbon and nitrogen for recovering tissues, so it increased under water stress levels. Water deficits induce dramatic increases in the proline concentration of phloem sap in medicinal and aromatic plants, suggesting that increased deposition of proline at the root apex in water stressed plants could in part occur via phloem transport of proline. A proline transporter gene, ProT2, is strongly induced by water and salt stress in Arabidopsis thaliana (Rentsch et al., 1996).
The maximum potassium content was in control (1.88%) followed by 20% water stress (1.75%) and minimum in 80% water stress (1.40%). Potassium content was affected by excessive water stress treatment, which resulted in the lowest percentage. These results are confirmed by those of Mirsa and Shrivastava (2000) for Japanese mint plants, Khalid (2001) for Nigella sativa L. plants and Hendawy and Khalid (2005) for Salvia officinalis L. plants. The maximum plumbagin content was in 80% water stress (0.2342%) and minimum in control (0.0984%). Lim et al., (2006) reported similar findings that ginsenoides content of Panax quinquefolium increased by the effect of water stress.
CONCLUSION
The various treatment combinations exhibited a significantly variability in morph-physiological growth parameters, biochemical parameters and economy yield attributing parameters of chitrak. Out of five stress levels, 80% water stress has influenced root length, dry herbage, plumbagin, potassium and proline content. In control conditions the plant height, number of leaf, total leaf area, stomatal conductance, transpiration rate, photosynthesis, CO2 utilization, H2O utilization and chlorophyll were found to be maximum. The significant improvement in morph-physiological and
biochemical attributes expressed as superior yield
attributing parameter which resulted in maximum
economic yield of any crop.
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