PLANT SIGNALING & BEHAVIOR 2016, VOL. 11, NO. 5, e1150400 (9 pages) http://dx.doi.org/10.1080/15592324.2016.1150400

RESEARCH PAPER

Ocimum sanctum leaf extract induces drought stress tolerance in rice Veena Pandeya, M.W. Ansarib, Suresh Tulac, R.K. Sahooc, Gurdeep Bainsa, J. Kumard, Narendra Tutejae, and Alok Shuklaa a Department of Plant Physiology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India; bDepartment of Botany, Zakir Husain Delhi College, Jawahar Lal Nehru Marg, New Delhi, India; cPlant Molecular Biology Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India; dDepartment of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India; e Amity Institute of Microbial Technology, Amity University, Noida, UP, India

ABSTRACT

ARTICLE HISTORY

Ocimum leaves are highly enriched in antioxidant components. Thus, its leaf extract, if applied in plants, is believed to efficiently scavenge ROS, thereby preventing oxidative damage under drought stress. Thus, the present study was performed in kharif 2013 and rabi 2014 season to evaluate the effect of aqueous leaf extract of Ocimum sanctum against drought stress in 2 rice genotype under glass house conditions. Here we show that various morpho- physiological (chlorophyll fluorescence, leaf rolling score, leaf tip burn, number of senesced leaves and total dry matter) and biochemical parameters (proline, malondialdehyde and superoxide dismutase content) were amended by Ocimum treatment in both the seasons. Application of Ocimum extract increased expression of dehydrin genes, while reducing expression of aquaporin genes in drought stressed rice plant. Thus, application of Ocimum leaf extract under drought stress can be suggested as a promising strategy to mitigate drought stress in economical, accessible and ecofriendly manner.

Received 18 December 2015 Revised 29 January 2016 Accepted 29 January 2016 KEYWORDS

Aquaporin; chlorophyll fluorescence; dehydrin; gene expression; leaf rolling; leaf senescence

Abbreviations: DHN, Dehydrin; MDA, Malondialdehyde; ROS, Reactive oxygen species; SOD, Superoxide dismutase;

TDM, Total dry matter

Introduction Ocimum is an aromatic plant in the family Lamiaceae. O. sanctum L., O. gratissium, O. canum, O. basilicum, O. kilimandscharicum, O. ammericanum, O. camphora, O. minimum L., O. tenuiflorum L. and O. micranthum are some of the commonly known species of genus Ocimum which grow in different parts of the world.1 Ocimum sanctum L. is found throughout India and is widely cultivated in homes and temple gardens. Apart from religious significance, it has a long history of medicinal use and is mentioned in Charak Samhita, the ancient textbook of Ayurveda.2 Fresh leaves and stems of Ocimum sanctum contain cirsilineol, cirsimaritin, isothymusin, isothymonin, apigenin, rosmarinic acid and eugenol.3 The other main chemical constituents of Ocimum include oleanolic acid, ursolic acid, carvacrol, linalool and b-caryophyllene.4 Drought is a major challenge limiting rice production. A common effect of drought stress is the generation of excess reactive oxygen species (ROS), which causes peroxidation of lipids, denaturation of proteins, mutation of DNA, disrupt cellular hemeostasis and various types of cellular oxidative damage, thereby affecting plant performance and yield.5 The ROS can be scavenged by a complex antioxidant system comprising of the non-enzymatic (ascorbate (AsA) and reduced glutathione (GSH)) and enzymatic antioxidants (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase

CONTACT Veena Pandey [email protected] Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis Group, LLC

(MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)). Thus, applying exogenous antioxidants or any strategy that can enhance antioxidant component in plants can be considered as a potent strategy for reducing oxidative stress and to ameliorate drought stress tolerance. Several reports showed that Ocimum is a rich source of antioxidants. The antioxidant property of any plant includes their secondary metabolites such as: flavonoids and phenolic acids. The methanolic leaf extract of Ocimum sanctum had high antioxidant potential and free radical scavenging properties.6 It was reported that the total phenolic content as well as radical scavenging activity was higher in leaf extract as compared to stem, which was due to high amount of carotenoids and ascorbic acid content.7 The antioxidant and free radical scavenging activity of aqueous ethanolic (1:1) extract of Ocimum sanctum was evaluated by means of DPPH radical, nitric oxide radical, superoxide anion radical and hydroxyl radical scavenging assays and it was suggested that some compounds in Ocimum sanctum are both electron donors and could react with free radicals to convert them into more stable products and to terminate radical chain reactions.8 Thus they concluded that Ocimum is a potential source of natural antioxidant. The antioxidant activities and total phenolics of methanolic and ethanolic leaf extracts of Ocimum sanctum was evaluated and it was observed that the ethanol: water extract had highest antioxidant activity,9 while the methanol extract possesses higher amounts

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of plant phenol10 which are also known to be responsible for the antioxidant activity of most of the plants. The antioxidant activity8 and reducing power11 of aqueous extract of leaves was found to be increased in a dose dependent manner. The total antioxidant capacity of Ocimum sanctum leaf extracts was investigated by measuring its ability to reduce the molybdate (Mo) ion, thereby depicting their capacity to reduce oxidative stress.12 Thus, our hypothesis was based on the fact that applying Ocimum leaf extract having well enriched antioxidant system in drought-stressed rice, may efficiently scavenge ROS, thereby preventing oxidative damage in rice and improving plant performance under drought stress. The methanolic and aqueous leaf extracts of O. gratissimum contains tannins, steroids, phenolics, terpenoids, flavonoids and cardiac glycosides which accounts for most of the antioxidant activity of plants. Anthraquinones were detected only in the aqueous extract while alkaloids were detected only in the methanolic extract.13 Ocimum leaf extract is rich in polyphenolic compounds and scavenged the radicals in a concentration dependent manner. The high concentrations of total phenolics, flavonoids and appreciable amounts of flavonols were present in the extract of O. americanum leaves and possess the ability to inhibit oxidative stress by antioxidant mechanisms.14 O. sanctum leaf extract possess free radical scavenging activity and contain alkaloids, tannins, flavonoids, and saponins.15 Chemical composition analysis of essential oil isolated from the leaves of Ocimum canum Sims. reported the presence of 36 compounds, among them camphor (39.77%) was the major compound. Several studies reported that high concentrations of bioactive monoterpenes like camphor led to irreversible damage of the whole plant, but low concentration or short term fumigations with camphor can strengthen the plant fitness as evidenced by altered gene expression of defence related genes like MAP3 kinase, ABF4 and RD29B.60 It was suggested that antioxidant activity of the O. canum essential oil might be due to the presence of mono and sesquiterpenes.16 Thus, unravelling the potential of Ocimum to induce drought stress tolerance in rice could be of great importance under increasing drought incidences. This might provide efficient ROS scavenging system and prevent cell damage under stress. To the best of our knowledge, there is no published literature reporting the efficacy of Ocimum sanctum leaf extract to mitigate drought stress in plants. So an attempt has been made to investigate the potential of Ocimum leaf extract to induce morpho- physiological and molecular changes in rice crop, leading to drought stress tolerance in rice.

Results Morpho-physiological analysis Ocimum treatment significantly amended various morphophysiological parameters like chlorophyll fluorescence (Fv/Fm), leaf rolling score, leaf tip burn, number of senesced leaves and total dry matter of drought-stressed rice leaves during both seasons as presented inTable 1. High chlorophyll fluorescence was maintained by Ocimum treatment under drought stress in both the rice genotypes, as compared to control. During kharif season, Ocimum treatment

resulted in highest fluorescence in PSD-17 (0.75), followed by PB-1 (0.74). During rabi season also, highest fluorescence was found in PSD-17 (0.72), followed by PB-1 in Ocimum treated plants. Leaf rolling was found to be more pronounced in treated plants as compared to untreated control during both growing seasons. During kharif season, Ocimum treatment resulted in compactly rolled leaf in PSD-17 (4.33), followed by PB-1 (2.67). During rabi season, Ocimum treated leaves of PSD-17 and PB-1 were equally rolled (3.33). Drought stress caused leaf tip burn in both rice genotypes during both the seasons, while Ocimum treatment significantly reduced incidence of tip burn except PSD-17 during kharif season where leaf tip burn was recorded more than control. PSD17 showed increase in leaf tip burn incidence under Ocimum treatment (13.43%) as compared to untreated control (8.35%) during kharif season; while during rabi season, tip burn was significantly reduced from untreated control (17.97%) to Ocimum treated plants (12.28%). In PB-1, during kharif season, untreated control showed 16.5% tip burn, which was reduced to 10.39% by Ocimum treatment; while during rabi season, untreated control showed 18.72% tip burn, which was reduced to 14.23% by Ocimum treatment. Ocimum treatment significantly decreased the number of senesced leaves in both rice genotypes during both growing seasons, compared with the control. In PSD-17, during kharif season, percent of senesced leaves per hill in untreated control and Ocimum-treated plants were 74.51% and 33.68% respectively; while during rabi season, it was 73.67% and 42.56% respectively. In PB-1, during kharif season, percent of senesced leaves per hill in untreated control and Ocimum-treated plants were 81.45% and 36.57% respectively; while during rabi season, it was 77.32% and 39.01% respectively. Total dry matter (TDM) per hill after harvesting was significantly higher in Ocimum treated plants as compared to untreated control in both the rice genotypes. During kharif season, Ocimum treatment resulted in highest TDM in PSD-17 (38.33g) followed by PB-1 (31.33g). During rabi season also, Ocimum treatment resulted in highest TDM in PSD-17 (28.67g) followed by PB-1 (25.33g).

Biochemical and yield analysis Table 2 shows the effects of Ocimum treatment on MDA, proline and SOD activity in drought-stressed rice leaves during kharif 2013 and rabi 2014 seasons. Ocimum treatment significantly decreased the concentration of MDA in all rice genotypes during both kharif and rabi growing seasons, compared with the control. The highest decrease in MDA content in Ocimum treated rice leaf during kharif season was observed in PSD-17 (19.3%) followed by PB-1 (16.4%) as compared to untreated control. During rabi season, the highest decrease in MDA content by Ocimum treatment was observed in PSD-17 (23.3%) followed by PB-1 (21.4%), compared with control. Seasonal variation was also evident from the MDA contents as average MDA contents were comparatively higher in the kharif rice as compared with the rabi season rice under drought stress.

PLANT SIGNALING & BEHAVIOR

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Table 1. Effect of Ocimum treatment on morpho-physiological attributes of 2 rice genotypes during kharif and rabi seasons. Kharif season 2013 Chlorophyll fluorescence (Fv/Fm)

Leaf rolling

Control

PSD-17 PB-1

0.75 § 0.002 0.74 § 0.003 Treatment (T) 0.0058 0.0169

0.70 § 0.019 0.71 § 0.007 Variety (V) 0.0045 0.0131

4.33 § 0.33 2.67 § 0.33 13.43 § 0.65 8.35 § 0.54 33.68 § 1.36 74.51 § 3.11 38.33 § 1.45 22.33 § 1.33 2.67 § 0.33 1.67 § 0.33 10.39 § 0.64 16.50 § 0.80 36.57 § 9.36 81.45 § 5.17 31.33 § 0.88 28.33 § 2.96 Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) 0.13 0.10 0.54 0.42 2.45 1.90 1.32 1.03 0.38 0.29 1.57 1.21 7.12 5.51 3.85 2.98 Rabi season 2014

0.72 § 0.01 0.71 § 0.00 Treatment (T) .004 .014

0.70 § 0.01 0.69 § 0.01 Variety (V) .003 .010

3.33 § 0.67 2.33 § 0.33 12.28 § 3.71 17.97 § 2.38 42.56 § 5.04 73.67 § 6.31 28.67 § 1.20 20.67 § 1.33 3.33 § 0.33 2.00 § 0.00 14.23 § 1.98 18.72 § 3.71 39.01 § 4.09 77.32 § 6.90 25.33 § 2.33 24.00 § 2.65 Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) .227 .176 1.96 1.52 2.57 1.99 1.68 1.30 .657 .509 5.67 4.39 7.44 5.76 4.85 3.76

S.Em. § CD at 5%

Treatment

Ocimum treatment significantly reduced the proline content for both the genotypes during both growing seasons, compared with the control. The highest reduction in proline content by Ocimum treatment during kharif season was observed in PB-1 (31.6%) followed by PSD-17 (22.7%), as compared to untreated control. During rabi season, the highest reduction in proline content in Ocimum treated rice leaf was observed in PSD-17 (53.1%), followed by PB-1 (35.8%), compared with control. Among both the seasons, more proline under drought stress was accumulated in control plants during rabi season as compared to kharif season, indicating that the rabi season rice experienced more severe drought. The Ocimum treatment significantly increased the activity of SOD in rice flag leaves for both the genotypes during both growing seasons, compared with the control. The highest increase in SOD activity by Ocimum treatment was observed in PB-1 (144.2%) followed by PSD-17 (97%) under kharif season, as compared to control. During rabi season, the highest increase in SOD activity in treated plants was observed in PSD17 (57.3%) followed by PB-1 (43%). Thus, among both the seasons, higher increase in SOD activity in all the genotypes was observed during kharif season. Fetching greater harvestable yield is the ultimate purpose of any strategy used to provide stress tolerance. In our study, Ocimum treatment significantly maintained higher number of

Control

Treatment

Control

TDM at maturity (g/hill)

Treatment

PSD-17 PB-1

Control

Senesced leaves per hill (%)

Variety

S.Em. § CD at 5%

Treatment

Leaf tip burn per hill (%)

Treatment

Control

filled grains as compared to untreated control plants, under drought stress during both the seasons. The highest increase in number of grain by Ocimum treatment was observed in PSD17 (45.4%) followed by PB-1 (8.8%) during kharif season, as compared to control. During rabi season, the highest increase in number of grain in treated plants was observed in PSD-17 (43.2%) followed by PB-1 (22.8%). Genotypic variation was also evident during both the seasons, under control as well as Ocimum treatment. Molecular analysis In this study, expression of 4 out of the 8 dehydrins and 2 out of the 37 aquaporin genes studied were significantly altered during drought stress. All the rice genotypes showed a significant increase in the transcript levels of dehydrins (DHN1, DHN2, DHN3 and DHN4) and decrease in the transcript levels of aquaporin genes (AQP1 and AQP2) under Ocimum treatment, as presented in Figs. 1 and 2. During both the seasons, in untreated control, there was no significant difference in the expression of dehydrin genes among the genotypes. But the extent of their increase under Ocimum treatment differs considerably in different genotypes. Highest normalized fold change in the expression of DHD1, DHD3, DHD4 was observed in PB-1, while expression of

Table 2. Effect of Ocimum treatment on biochemical and yield attributes of 2 rice genotypes during kharif and rabi seasons. Kharif season 2013 MDA (mg/g FW)

Proline (mmol/g FW)

Variety

Treatment

Control

Treatment

PSD-17 PB-1

3.41 § 0.12 3.75 § 0.05 Treatment (T) 0.073 0.213

4.23 § 0.23 4.49 § 0.13 Variety (V) 0.057 0.165

2.75 § 0.38 2.74 § 0.21 Treatment (T) 0.097 0.281

3.08 § 0.05 3.33 § 0.06 Treatment (T) .058 .169

4.02 § 0.04 4.24 § 0.10 Variety (V) .045 .130

2.19 § 0.02 2.60 § 0.01 Treatment (T) .077 .223

S.Em. § CD at 5% PSD-17 PB-1 S.Em. § CD at 5%

Control

SOD (U/mg protein) Treatment

Number of filled grains per panicle

Control

Treatment

Control

3.56 § 0.03 2.68 § 0.08 4.01 § 0.09 2.98 § 0.19 Variety (V) Treatment (T) 0.075 0.094 0.217 0.272 Rabi season 2014

1.36 § 0.10 1.22 § 0.10 Variety (V) 0.072 0.211

100.33 § 2.03 86.33 § 3.48 Treatment (T) 2.448 7.073

69.00 § 1.15 79.33 § 2.67 Variety (V) 1.896 5.478

4.67 § 0.02 4.05 § 0.04 Variety (V) .060 .173

1.78 § 0.15 2.00 § 0.08 Variety (V) .054 .154

96.00 § 6.03 96.67.33 § 2.19 Treatment (T) 2.207 6.376

67.00 § 0.58 78.67 § 3.76 Variety (V) 1.710 4.939

2.80 § 0.03 2.86 § 0.14 Treatment (T) .070 .204

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Figure 1. Effect of Ocimum treatment on expression of 4 dehydrin genes on drought stressed rice during (A) kharif and (B) rabi seasons.

Figure 2. Effect of Ocimum treatment on expression of 2 aquaporin genes on drought stressed rice during kharif and rabi seasons.

PLANT SIGNALING & BEHAVIOR

DHD2 was highest in PSD-17 (Fig. 1). Normalized fold change in the expression of aquaporin genes decreased considerably under Ocimum treatment in both the seasons, as compared to control. Highest reduction in normalized fold change in the expression of AQP1 and AQP2 was observed in PB-1 genotype, in both the seasons. (Fig. 2).

Discussion The use of plant extracts and phyto-products to alleviate various stresses is nowadays gaining attention due to their availability, cost effectiveness and biodegradability. In the present study, the effect of Ocimum leaf extract on different drought related parameters in 2 rice genotypes was evaluated. Previous reports showed that drought stress reduces chlorophyll fluorescence (Fv/Fm) in different rice cultivars.17-19 In our study, Ocimum treatment significantly maintained the Fv/Fm values under drought stress, while the fluorescence was found lowest in untreated control in both rice genotypes, during both the seasons. Leaf rolling is one of the acclimation responses of rice and helps in maintaining internal plant water status.5 Ocimum treatment resulted in compactly rolled leaf as compared to untreated control plants. Ocimum treated PSD-17 genotype showed highest rolling score during both the season, as compared to PB-1, thus preventing large amount of transpirational water loss under drought stress. The present study indicated that drought tolerance of rice genotypes was enhanced by Ocimum treatment as evidenced by reduced leaf tip burn and drought-induced senescence. Percent of senesced leaves per hill was lowest in Ocimum treated PSD17 during kharif season, while during rabi season, it was lowest in Ocimum treated PB-1. Seasonal variation was also evident from the percent of leaf tip burn and senesced leaf and these parameters were comparatively higher in the rabi season rice as compared to kharif season rice, indicating that negative impact of drought stress was more severe during rabi season. Reports revealed that total dry matter (TDM) production decreased with decreasing soil moisture level.20,21 Ocimum treatment significantly found to maintain higher TDM after harvesting under drought stress, as compared to control. Seasonal variation in TDM was also evident in both the varieties and lower TDM was recorded during rabi season. ROS production is inevitable consequence of drought stress and severely damage cell component resulting in reduced performance of plant. Ocimum leaves are well known for their antioxidant potential and efficiently scavenge free radicals.6-9 Prominent antioxidant mechanism ensures low lipid peroxidation resulting in low MDA content under drought stress. In our study, Ocimum treatment significantly reduced MDA content in flag leaf of drought stressed rice as compared to untreated control in both the seasons. Lowest amount of MDA was found in PSD-17 during kharif and rabi season in Ocimum treated plant, as compared to control. Ursolic acid, a triterpene, isolated from O. sanctum has been shown to be effective in protecting against lipid peroxidation.22 Leaf extract of O. sanctum was found to inhibit lipid peroxidation in rats intoxicated with arsenic23 and diazinon.24 Ethanolic leaf extract of O. basilicum decreased the lipid peroxidation concentration in parasitized mice.25

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Changes in the concentration of proline have been observed in many studies on drought stressed rice.26-28Proline accumulation may be considered as an indicator of drought susceptibility. Rice plants which showed more tolerance under drought stress, accumulated less proline than sensitive ones.29 Our results showed that Ocimum treatment significantly reduced proline content in drought stressed rice leaf. Lowest proline content was found in PB-1 during kharif season, while during rabi season, lowest proline was noted in PSD-17. Superoxide dismutase (SOD) is an important antioxidant enzyme which protects the plant with excessive superoxide radicals formed in plants under stress. Ocimum treatment significantly increased SOD activity in drought stressed rice leaf as compared to untreated control, in both the seasons. Ocimum treatment resulted in highest SOD activity in PB-1 during kharif and rabi season. The O. sanctum leaves were found to have antioxidant enzymes like SOD, CAT, GPX, GSH and ascorbate and the extract was found to have potent antioxidant activity.30 Administration of hydro-alcoholic extract of O. sanctum leaves increased SOD activities in rats intoxicated with arsenic.23 O. basilicum leaf extract increased antioxidant enzymes namely SOD and CAT in rats.24 Ethanolic leaf extract of O. basilicum increased the activities of SOD, CAT and GPX in Plasmodium parasitized mice, indicating that the extract scavenged superoxide radicals in mice.25 Study on the antioxidant effect of O. basilicum aganist gibberllic acid and auxin supplemention in broilers ration showed that antioxidant activity (SOD, CAT, GSH, GPX) significantly increased when O. basilicum is added.31 Foliar treatment of tomato plants with 30% aqueous extract of O. sanctum increased the antioxidant enzymes.32 Drought triggers a variety of response at molecular level, by modifying a range of genes expression. Among various genes, aquaporins and dehydrins play a major role in providing drought tolerance in plants. Aquaporins (AQP) are known to facilitate water movement across membrane,33,34 while dehydrins (DHN) or group 2 LEA proteins are thought to be involved in water binding molecules and stabilizing both macromolecules and membranes.35 In our study, the gene expression of 4 dehydrin genes were found to be increased significantly by Ocimum treatment while expression of 2 aquaporin genes were found to be reduced significantly by Ocimum treatment. Moreover, we can say some elicitors must be present in Ocimum extract which triggered signaling cascade in rice plants that led to altered expression of aquaporins and dehydrins. Like our results, a positive correlation of dehydrin expression with drought tolerance has been reported in many studies.36-39 More recently, study in tomato reported that overexpression of dehydrin tas14 gene helps in improving the drought stress tolerance.40 The overexpression of OsDhn1 in transgenic rice plants enhanced drought stress tolerance as judged by chlorophyll fluorescence (Fv/Fm), fresh and dry weight, water and chlorophyll content, and suggested that OsDhn1 plays an important role in the stress tolerance via scavenging reactive oxygen species (ROS).41 Several reports supporting the negative role of aquaporins under drought stress are available. Over-expressing plasma membrane aquaporin AtPIP1b in transgenic tobacco had a

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negative impact during drought stress, with the transgenic plants wilting more rapidly than the wild type plants.42 Thus it was suggested that a general increase in water transport in most plant tissues and cells is harmful under drought stress. Transgenic tobacco and Arabidopsis plants over-expressing PIP1;4 or PIP2;5 showed rapid water loss under dehydration stress and it was speculated that enhanced water transport via the plasma membranes of cells may be deleterious under water stress.43 Constitutive over-expression of an AQP gene GoPIP1 from G. orientalis in Arabidopsis led to decreased drought resistance and enhanced drought sensitivity in transgenic Arabidopsis. The transgenic plants over-expressing GoPIP1 had lower leaf water content and lower water potential and had faster water loss through leaves to the atmosphere, indicating that GoPIP1 might affect stomatal aperture thus modifying water movement.44 In conclusion, the present study demonstrates the efficacy of Ocimum leaf extract against drought stress in different rice genotypes. It was observed that spraying Ocimum leaf extract increased SOD activity and decreased accumulation of MDA and proline in drought stressed flag leaves of rice, implying that plants have had experienced less stress. Moreover, the percent of senesced leaf and tip burn incidences were reduced by Ocimum treatment. The application of O. sanctum leaf extract led to alteration in gene expression of water channel proteins aquaporins and defense related LEA proteins like dehydrins and amended the biochemical processes in rice under drought stress, which ultimately provided plant to tolerate stress for more period of time. This alteration in gene expression might have occurred

due to certain elicitor molecules which are expected to be present in Ocimum leaf extract and induced a series of signaling cascade in rice, resulting better adaptation by rice under stress (Fig. 3). Aqueous extracts of O. gratissimum leaves induced the production of phytoalexins in soybean cotyledons and sorghum mesocotyls and also induced systemic resistance in cucumber as reflected by increase in chitinase production.45 Aqueous extract of O. basilicum was found to induce the expression of extracellular signal-regulated kinase 2 (ERK2), which is capable of regulating several transcription factors, thus suggesting that the extract can mediate the ERK2 MAP-kinase signal pathway.46 Further experiments are necessary to determine the best time of application and concentration of Ocimum leaf extract. Of equal importance is the elucidation of the extract components responsible and the signaling mechanism involved.

Materials and methods Experimental site Glass house experiments were conducted in the kharif season (June to November) 2013 and repeated in the rabi season (February to July) 2014 at Center of Advance Studies, Department of Plant Pathology, College of Agriculture, G.B.P.U.A.T., Pantnagar. Geographically, the site lies in Tarai plains about 30 km southwards of foothills of Shivalik range of the Himalayas at 29 N latitude, 79 290 E longitude and at an altitude of 243.8 meter above the mean sea level. Treatments were arranged in a completely randomized design with 4 replications.

Figure 3. Ocimum induced various responses in drought stressed rice plants and hypothetical signal transduction pathway. SOD- Superoxide dismutase, MDA- Malondialdehyde, TDM- Total dry matter.

PLANT SIGNALING & BEHAVIOR

Preparation of O. sanctum leaf extract The leaves of O. sanctum were collected from local area at Pantnagar, Uttarakhand. Fresh plant leaves of O. sanctum were washed under running tap water, air dried and then homogenized to fine powder. 20 gm. of air-dried powder of leaves was added to 300 ml distilled water. It was then boiled on slow heat for 24 h and then filtered. The filtrate concentrated to make the final volume one by third of the original volume with the help of water bath and this 20% (w/v) concentrated solution was used as Ocimum extract. Drought stress and Ocimum treatment The seeds of rice genotypes, namely, Pusa Basmati 1(PB 1) and Pant Sugandh Dhan 17 (PSD 17) were obtained from Seed Production Center, G.B.P.U.A.T., Pantnagar. Seeds of both rice genotypes were sown in nursery and transplanted after 21 days in 5 kg capacity pots having sterilized soil. The pots were kept in the well-ventilated glasshouse during the whole growing seasons. Four replications were taken and the number of plants per pot was maintained as four. Pots containing plants of each genotype were divided into 2 groups. One set was given Ocimum treatment under drought stress, while other set was not sprayed with Ocimum and maintained as control. Drought stress treatment was induced at the time of flowering by irrigation withholding for 5 subsequent days in all the pots. On second day of drought application, aqueous extract of O. sanctum was sprayed on all the pots by using hand held plastic spray bottle, except control. The soil moisture content in pots on fifth day was recorded as 11§2% and 8§2% during kharif 2013 and rabi 2014 season respectively. 1.Morpho-physiological assay: Chlorophyll fluorescence (Fv/Fm), leaf rolling score, tip burn (% per hill), number of senesced leaves after drought (% per hill), total dry matter (g) Morpho-physiological parameters like chlorophyll fluorescence and leaf rolling were observed on third day of stress while observations on leaf tip burn, number of senesced leaves per hill was taken on the fifth day of drought application. Chlorophyll fluorescence of rice flag leaves was measured with the help of Handy PEA (Hansatech, UK). Dark adaptation of samples was achieved via lightweight plastic leaf clips, the leaf clip shutter blade was closed to prevent the entry of light and the clip left in place for 10 minutes to provide dark adaptation. To perform a measurement, the Handy PEA sensor unit was held over the clip and the shutter opened. A single buttonpress immediately displayed automatically calculated fluorescence parameters F0, Fm, and Fv/Fm. All the observations were recorded in the forenoon hrs (9–11 AM) to avoid photoinhibition. Leaf rolling may be a useful strategy to maintain leaf water potential in rice under drought. It was scored visually after 2 days of the drought treatment using scales of 0–5 with 1 being the first evidence of rolling and 5 being a closed cylinder. Leaf tip burning and leaf senescence on fifth day of drought stress was observed and calculated as the cumulative percentage of

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leaf tip browning and leaf dying respectively, out of the total leaf population. The total plant dry matter per plant (g) was recorded at maturity stage by uprooting the complete plant and then placing the plant sample in the oven at 65 C for 3 days. Biochemical assay: Proline content, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity All the biochemical analysis was performed on the fifth day of drought application. Proline content was determined following the method of Bates et al.47 Leaf tissue (0.2 g) was homogenized in 4 ml of sulphosalicylic acid (3%) and centrifuged at 10,000 g for 30 min. About 2 ml of the supernatant was taken in a test tube and to it 2 ml of glacial acetic acid and 2 ml ninhydrin reagent were added. The reaction mixture was boiled in water bath at 100 C for 30 min. After cooling the reaction mixture, 4 ml of toluene was added and vortexed for 30s, the upper phase containing proline was measured with spectrophotometer at 520 nm using toluene as a blank. Lipid peroxidation in terms of total MDA concentration (mg/g FW) was estimated by the method of Heath and Packer.48 The extent of lipid peroxidation was evaluated by the thiobarbituric acid reaction. 0.1g of leaf was homogenized in 0.1% trichloroacetic acid (1:10, w:v) and centrifuged at 15,000 g for 10 min. 0.5 ml of the supernatant was incubated with 1.5 ml of 0.5% thiobarbituric acid diluted in 20% trichloroacetic acid at 95 C for 25 min and then cooled in ice bath. Measure the absorbance of the supernatant at 532 nm and 600 nm. The concentration of thiobarbituric acid reactive substances was calculated as malondialdehyde equivalent using the extinction coefficient (155 mM¡1 cm¡1). SOD activity was assayed by the method of Giannopolitis and Ries.49 The reaction mixture contained 1.3 mM riboflavin, 13 mM L- methionine, 0.05 M Na2CO3, (pH 10.2), 63 mM p– nitroblue tetrazolium chloride (NBT) and crude plant extract. Reaction was carried out under illumination (75 mmol photon m¡2 s¡1) from fluorescent lamp at 25 C. The initial rate of reaction as measured by the difference in increase in absorbance at 560 nm in the presence and absence of extract was proportional to the amount of enzyme. Molecular assay Sequence-retrieval and microarray-based expression study of drought-related dehydrin and aquaporin genes Sequence of drought-related dehydrin and aquaporin genes (Table S1) were retrieved from Rice Genome Database RGAP7 (http://rice.plantbiology.msu.edu/;50) using BLASTn. The retrieved sequences were confirmed by evaluating the annealing of primers used in the relevant studies by means of Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primerblast/). Further, the microarray probe set ids were retrieved via Rice Oligonucleotide Array Database (http://ricearray.org/;51). Among the 8 dehydrin genes and 37 aquaporins genes reported so far, specific probe set ids could be established for all. To examine the expression profile, publicly accessible data for single microarray platform – 51 K Affymetrix gene chip was exercised (https://www.genevestigator.com/gv/plant.jsp;52,53).

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Heat map with average linkage hierarchical clustering was produced with Multi Experiment Viewer software (http://www. tm4.org/mev/;54) by means of Pearson correlation as the distance metric. RNA isolation and quantitative real-time PCR (qRT-PCR) Leaf samples of drought stressed rice plants were used for RTPCR. Total RNA was isolated via TriZOL LS reagent (Invitrogen, http://www.invitrogen.com) as per the manufacturer’s instructions, and poly(A)-RNA was isolated. It was used for making cDNA using the RevertAid H minus first-strand cDNA synthesis kit (Fermentas, http://www.thermoscientificbio.com/ fermentas). Expression analysis of the dehydrin and aquaporin genes was performed by qRT-PCR.55 The relative levels of the transcripts accumulated for dehydrin and aquaporin genes were normalized to a–tubulin (primers detailed in Table S2) and the dehydrin and aquaporin genes expression in rice plant56 using the 2–DDCt method from 3 independent experiments.57 The PCR proficiency, which is reliant on the assay, performance of the master mix and quality of the sample, was calculated as efficiency D 10 (–1/slope) – 1 (3.6C slope C 3.1) by the software itself (Applied Biosystems, http://www.applied biosystems.com) ‘C’ is defined as threshold cycle. Statistical analysis Data presented are the averages of 3 replicates. The experiment was performed in a factorial completely randomized design. The data from the experiments were subjected to 2-way ANOVA and followed by separation of means at P  0.05. Standard error of each mean was calculated to represent that in the tables.

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

Acknowledgments The first author is highly thankful to the Department of Science & Technology, New Delhi, India for providing financial support. The authors are also grateful to All India Coordinated Rice Improvement Project, Indian Council of Agricultural Research, New Delhi, India for providing necessary facilities for conducting the present investigation.

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