The effect of brassinolide on the activities of certain enzymes like IAA oxidase, protease and ribonuclease of two sorghum varieties (â€˜CSH-5â€™ and â€˜CSH-6â€™) grown in four saline experimental sites of Karaikal viz. Varchikudy, Pogalam, Kilavur andÂ  Mallavur, was studied. Brassinolide-treatment resulted in lowered IAA oxidase, protease and ribonuclease activities. The study revealed that brassinolide was more effective in lowering the enzyme activities in Mallavur than other saline sites (Pogalam, Kilavur and Varchikudy).Â

Vidya Vardhini, B

Journal of Phytology

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Journal of Phytology 2012, 4(2): 30-33                                                                                ISSN: 2075-6240  Available Online: http://journal-phytology.com/    Effect of brassinolide on certain enzymes of sorghum grown in saline soils of Karaikal  B. Vidya Vardhini  Department of Botany, Telangana University, Nizamabad-503175, Andhra Pradesh, India, India  Abstract  The effect of brassinolide on the activities of certain enzymes like IAA oxidase, protease and ribonuclease of two sorghum varieties (‘CSH-5’ and ‘CSH-6’) grown in four saline experimental sites of Karaikal viz. Varchikudy, Pogalam, Kilavur and  Mallavur, was studied. Brassinolide-treatment resulted in lowered IAA oxidase, protease and ribonuclease activities. The study revealed that brassinolide was more effective in lowering the enzyme activities in Mallavur than other saline sites (Pogalam, Kilavur and Varchikudy).   Keywords: Brassinolide, IAA oxidase , protease , ribonuclease, sorghum   INTRODUCTION       Plant growth regulators play a very positive role in stress alleviation of various plants [1] Brassinosteroids (BRs) are a novel group of phytohormones with significant growth promoting nature [2] and are ubiquitous in plant kingdom as they were reported in all plants tested (alga, bryophyte, pteridophyte, gymnosperms, dicots and monocots) [3] . BRs are considered as growth regulators with pleiotropic effects, as they influence diverse physiological processes like growth, germination of seeds, rhizogenesis, senescence etc. and also confer resistance to plants against biotic and abiotic stresses [4]. BRs confer tolerance to a wide range of abiotic stresses in Arabidopsis thaliana and Brassica napus [5]. Krishna [6] also reported that BRs play a very prominent role in reducing the biotic and abiotic stresses in plants. In case of Eucalyptus camaldulensis, treatment of seeds with 24-epibrassinolide resulted in increased seed germination under saline conditions [7].  BRs increased the tolerance to high temperature in brome grass, where it has also been demonstrated that the tolerance in plants to high temperature due to the application of BRs is being associated with induction of de novo polypeptide protein (heat shock protein) synthesis [8]. Brassinolide was found to alleviate the negative impact of saline stress by monitoring the antioxidative system of two varieties of sorghum plants grown in two different saline soil of Karaikal [9]. Thus Xia et al. [10] aptly stated that BRs induce plant tolerance to a wide spectrum of stresses.      Sorghum vulgare Pers. is one of the five major cereal crops widely grown in the tropical and sub tropical parts of the world. It is the staple food for a large number of people and also a main source of fodder, feed and industrial raw material. It is a rain fed crop and poor monsoon and extended dry conditions play a devastating influence on the crop performance [11]      Malibari et al. [12] reported that salinity stress is one of the critical environmental stresses that affect that ultimate yield of crops all around the world. Salinity usually effects the dry matter production, ionic relations, metabolic variations, physiological processes and water contents of the soil which leads to reduced growth of the plants. Karaikal is a part of the Union Territory of Puducherry. It falls in the Nagapatinum district of Tamil Nadu and lies in the east coastal belt of Bay of Bengal which usually experiences erratic rain fall. The Tsunami of 2004 caused many changes in the already poor soil texture of the land. After this massive deluge, both soil and water sources were highly enriched with salts of different chemical nature. The coastal soils have turned out to be more saline due to the process of secondary salinisation. The saline water consists of excess of neutral soluble salts mostly chlorides and sulfates of Na, Ca, and Mg. The present study was done to find out the effect of brassinolide, a potential plant growth regulator on activities of certain enzymes like IAA oxidase, protease and ribonuclease of sorghum plants grown in four experimental saline sites viz., Varichikudy, Pogalam, Kilavur and Mallavur of Karaikal, a tsunami hit area of Puducherry Union Territory of India.  MATERIALS AND METHODS Chemicals and plant material       Brassinolide (double) is a commercially available brassinosteroid which is manufactured by Bahar Agrochem & Feeds Pvt. Ltd, Ratnagiri, Maharastra, India, Ltd. and is marketed by Godrej Agrovet Ltd., Hyderabad, Andhra Pradesh, India. Brassinolide (Double) consists of 0.1% of brassinolide, 2.0% of emulsifier and 97.9 % of solvent IPA.       Seeds of sorghum (Sorghum vulgare. Pers) varieties ‘CSH-5’ and ‘CSH-6’ were purchased from National Seeds Corporation, Coimbature, India. CSH-5 is a hybrid variety of 2077A × CS3541 and is a kharif crop (sown in early summer for harvesting in autumn). CSH-6 is a hybrid variety of 2219A × CS3541 and is also a kharif (sown in early summer for harvesting in autumn) crop.      The seeds were sown in earthen pots containing 10 kg of saline soil (collected from four different sites viz. Varichikudy, Pogalam, Kilavur and Mallavur of Karaikal) and compost in a 10: 1   Received: Dec 15, 2011; Revised: Jan 10, 2012; Accepted: Feb 15, 2012.  *Corresponding Author  B. Vidya Vardhini Department of Botany, Telangana University, Nizamabad-503175, Andhra Pradesh, India, India Tel: +91-9959173377; Fax: +91-841 222212 Email: drvidyavardhini@rediffmail.com    Journal of Phytology 2012, 4(2): 30-33  31ratio. Plants were grown in under natural day length. Brassinolide (double) was supplied to the plants as foliar spray at 2 different concentration levels viz., 2.0 and 3.0 µM on 35th, 45th and 55th DAS (days after sowing). Two sets of water treated controls were also maintained. On 70th DAS, enzyme studies were conducted. The leaves were harvested in the early hours of morning, washed with distilled water, the surface water was blotted off and the leaves were kept in an ice-box, which were later used for enzyme studies.  IAA oxidase (E.C.1.11.1.8)       IAA oxidase (IAAO) was extracted by the method of Hillman and Galastan [13]. Seedlings were homogenized in chilled phosphate buffer (pH = 6.1). The assay mixture contained IAA, enzyme extract and phosphate buffer. The reaction was terminated by adding 10% (w/v) trichloroacetic acid. The residual IAA was estimated by Salper reagent and quantified by the use of IAA standard graph.  Protease (E.C.3.4.22.44)       200mg of the seedlings was homogenized in a pre chilled mortar and pestle using 10ml of chilled 0.2M sodium acetate buffer( pH=5.2 The supernatant was used as enzyme extract and protease activity was estimated of the amount of protein present according to Lowry et al. [14] method.  Ribonuclease (E.C.3.1.27.5)       The seedlings was ground with potassium acetate buffer (pH = 6.5) and centrifuged. The supernatant was used as enzyme extract. Ribonuclease (RNase) activity was estimated by Corbishley et al. [15] method.      The values were presented as Mean ± S.E. of 5 replicates. ANOVA - one way revealed that the mean values of different activities of 28-homoBL are significant at 5% level of significance over control. The values were calculated employing SPSS 16.0 statistical software.  RESULTS        Brassinolide application resulted in reduction in the activity of the enzyme IAAO in the two varieties of sorghum plants (‘CSH-5’ and ‘CSH-6’) grown in all the four saline experimental sites (Varchikudy, Pogalam, Kilavur and Mallavur) of Karaikal compared to the control plants (Table 1). The present experiments revealed that the activity of IAAO extracted  from sorghum plants grown in saline soils of stressed but untreated controls was more in  Mallavur than Varichikudy, Pogalam and Kilavur in both the CSH-5 and CSH-6 varieties. But the application of brassinolide resulted in reduced activity of IAAO in Mallavur than Pogalam, Kilavur and Varichikudy in the two varieties of sorghum studied. The maximum reduced activity was recorded in plants treated with 3 µM brassinolide in both the varieties and in all the experimental saline soil sites of Karaikal.Table1. Effect of brassinolide on the activities of IAA oxidase, protease and ribonuclease of two varieties of sorghum grown in two experimental sites of Karaikal  Varieties /Experimental sites Treatments IAA oxidase activity*a Protease activity* b Ribonuclease activity* c CSH-5/ Varchikudy Control-I Control-II 2µM BL 3µM BL 3.19 ± 1.02 3.21 ± 1.11 2.66 ± 1.67 1.66 ± 1.10 08.46 ± 0. 08 08.00 ± 0.09 05.92 ± 0.08 04. 63 ± 0.02 0.626 ± 0.08 0.645 ± 0.10 0.354 ± 0.06 0.322 ± 0.02 CSH-5/ Pogalam Control-I Control-II 2µM BL 3µM BL 3.45 ± 1.02 3.87 ± 1.45 2.71 ± 1.81 1.98 ± 1.20 08.78 ± 0.06 07.34 ±0.09 05.70 ± 0.08 04.86 ± 0.08 0.676 ± 0.10 0.645 ± 0.12 0.474 ± 0.08 0.318 ± 0.04 CSH-5/ Kilavur Control-I Control-II 2µM BL 3µM BL 3.65 ±1.21 3.22 ±1.34 2.43 ± 1.35 1.59 ± 1.22 08.93 ± 0.09 08.02± 0. 06 06.21 ± 0.09 05.54 ± 0.10 0.706 ± 0.60 0.685 ± 0.12 0.414 ± 0.06 0.382 ± 0.07 CSH-5/ Mallavur Control-I Control-II 2µM BL 3µM BL 4.66 ±1.22 4.33 ±1.24 2.31 ±1.27 1.34 ±1.00 10.98 ± 0.05 09.86 ± 0.07 02.34 ± 0.09 02.10 ± 0.08 0.706 ± 0.09 0.685 ± 0.12 0.274 ± 0.04 0.212 ± 0.08 CSH-6/ Varchikudy Control-I Control-II 2µM BL 3µM BL 3.29 ± 1.12 3.41 ± 1.00 2.69 ± 1-08 2.00 ± 1.12 08.64 ± 0. 04 08.20 ± 0.07 05.72 ± 0.08 05. 03 ± 0.05 0.676 ± 0.09 0.655 ± 0.10 0.494 ± 0.02 0.402 ± 0.06 CSH-6/ Pogalam Control-I  Control-II 2µM BL 3µM BL 3.50 ± 1.22 3.61 ± 1.02 2.88 ± 1.08 2. 08 ± 1.12 09.98 ± 0.05 08.88 ± 0.08 06.34 ± 0.06 06.10 ± 0.04 0.686 ± 0.09 0.645 ± 0.12 0.474 ± 0.04 0.392 ± 0.08 CSH-6/ Kilavur Control-I Control-II 2µM BL 3µM BL 3.21 ± 1.22 3.32 ± 1.00 2.36 ± 1.00 1.95 ± 1.20 08.98 ± 0.05 08.86 ± 0.07 05.34 ± 0.08 04.10 ± 0.09 0.696 ±0.09 0.665 ± 0.10 0.484 ±0.04 0.462 ± 0.02 CSH-6/ Mallavur Control-I Control-II 2µM BL 3µM BL 4.70 ± 1.28 4. 42 ± 1.22 2.11 ± 1.02 1.26 ± 1.22 10.62 ± 0.10 09.16 ± 0.05 02.54 ± 0.01 02.20 ± 0.12 0.716 ± 0.08 0.705 ± 0.10 0.264 ± 0.02 0.222 ± 0.02          BL = Brassinolide          *The presented values are Mean ± SE. ANOVA one way classification revealed that the differences are significant at 5% level of significance.          a = IAA oxidase activity is expressed in terms of the amount of IAA destroyed in µg g-1Fr.Wt./20 min.          b = Protease activity is expressed in terms of the amount of protein destroyed in µg g-1Fr. Wt. /30 minutes.          c = RNase activity is expressed in absorbance units which indicated the amount of nucleotides formed due to depolymerization of RNA. Vidya Vardhini   32      Brassinolide application resulted in reduction in the activity of the enzyme protease in the two varieties of sorghum plants (‘CSH-5’ and ‘CSH-6’) grown in all four saline experimental sites (Varchikudy, Pogalam, Kilavur and Mallavur) of Karaikal compared to the control plants (Table 1). The present experiments revealed that the activity of protease extracted from sorghum plants, in the stressed but untreated controls was more in  Mallavur than Varichikudy, Pogalam and Kilavur in both the CSH-5 and CSH-6 varieties. But the application of brassinolide resulted in reduced activity of protease in Mallavur than Varichikudy, Pogalam and Kilavur in the two varieties of sorghum studied. The maximum reduced activity was recorded in plants treated with 3 µM brassinolide in both the varieties and in all the experimental saline soil sites of Karaikal.       Brassinolide application resulted in reduction in the activity of the enzyme RNase in the two varieties of sorghum plants (‘CSH-5’ and ‘CSH-6’) grown in all the saline experimental sites (Varchikudy, Pogalam, Kilavur and Mallavur) of Karaikal compared to the control plants (Table 1). The present experiments revealed that the activity of RNase extracted from sorghum plants, in the stressed but untreated controls was more in Mallavur than Varichikudy, Pogalam and Kilavur in both the CSH-5 and CSH-6 varieties. But the application of brassinosteroids resulted in reduced activity of RNase in Mallavur than Varichikudy, Pogalam and Kilavur in the two varieties of sorghum studied. The maximum reduced activity was recorded in plants treated with 3 µM brassinolide in both the varieties and in all experimental saline soil sites of Karaikal.   DISCUSSION       The activity of IAAO was decreased by the supplementation of BRs to four varieties of sorghum seedlings grown under PEG-imposed water stress [11]. Thus BRs seem exhibiting IAA-sparing influence. Similar decrease in the IAAO activity in the mung bean epicotyls treated with BL was observed by Wu and Zhao [16]. On the other hand the gravitropic curvature of maize primary roots caused by BL was found promotive in the presence of IAA indicating the interactions of auxins and BRs Kim et al. [17].       Seed treatment of BRs to the four varieties of sorghum seedlings grown under PEG-imposed water stress exhibited reduced protease activity. The supplementation of BRs to wheat plants [18] and rice [19] resulted in enhanced soluble proteins under various stress conditions. The decrease in the protease activity might have been due to reduced protein degradation and denovo polypeptide synthesis [19]. Further Vardhini et al. [20] reported decreased protease activity in sorghum seedlings [20] grown under PEG – imposed water stress.      Elevated activity of RNA polymerase and lowered activity of RNase and DNase were observed in mung bean seedlings when treated with epibrassinolide [21]. The present study with whole plant system also revealed reduced RNase activity in brassinosteroid treated sorghum plants grown in saline soils of Karaikal.  CONCLUSIONS       BRs bind to the membrane proteins and scavenge the reactive oxygen species which are generated by heavy metal toxicity, thereby reducing the membrane destruction that from AOS (active oxygen species)-induced oxidative damage [22]. After binding to the membrane proteins BRs may enhance the enzyme and metabolic activities, thus detoxifying heavy metals in plants. How ever further research is required to unravel the mechanism of BRs in alleviating the impact of abiotic stress in crop plants. Li et al. [66] emphatically stated that ‘Treatment of seedlings with BL may be a useful management tool for afforestation projects in arid and semiarid areas’.   ACKNOWLEDGEMENTS       The author thanks Prof. S. Seeta Ram Rao, Department of Botany, Osmania University for his critical suggestions in the paper. The financial support from University Grants Commission (UGC), New Delhi [MRP-655/05 (UGC – SERO); Link No 1655.0] India is gratefully acknowledged.  REFERENCES  [1] Vardhini B.V., S. Anuradha, E. Sujatha, and S.S.R. Rao. 2010. Role of Brassinosteroids in Alleviating Various Abiotic and Biotic Stresses - A Review. In: N. A. Anjum, (Ed.), Plant Nutrition and Abiotic Stress Tolerance I. Plant Stress 4 (Special Issue 1), London: Global Science Books, pp.56-61. [2] Vardhini, B.V., E. Sujatha, and S.S.R. Rao. 2011. Effect of brassinolide on biochemical composition of radish (Raphanus sativus). Bioinfolet 8:404-406 [3] Vardhini, B.V., S. Anuradha, and S.S.R.  Rao. 2006. Brassinosteroids-New class of plant hormone with potential to improve crop productivity. Indian J. Plant Physiol. 11: 1-12. [4] Bajguaz, A. 2009. Isolation and characterization of brassinosteroids from algal cultures of chlorella vulgaris Beijernick (Trebouxiophyceae). J. Plant Physiol. 166: 1946-1949. [5] Kagale, S., K. D . 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Effect of brassinolide on certain enzymes of sorghum grown in saline soils of Karaikal

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Effect of brassinolide on certain enzymes of sorghum grown in saline soils of Karaikal

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