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Physiological and Biochemical Analysis of Desiccation Tolerance in Eragrostiella Bifaria. Ramyashree C ,Banupriya T G , Sharathc

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Physiological and Biochemical Analysis of Desiccation Tolerance in Eragrostiella Bifaria. Ramyashree C ,Banupriya T G , Sharathc The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367 Physiological and Biochemical analysis of Desiccation Tolerance in Eragrostiella bifaria. Ramyashree c1,Banupriya T G1, Sharathchandra R.G1. 1Department of Studies and Research in Environmental Science, Tumkur University, Tumakuru. Corresponding author E-mail: [email protected] Telephone number: 8095502894 Acknowledgements:Thanks to Indo-French Centre for the Promotion of Advanced Research (IFCPRA) and Directorate of Minorities, KarnatakaGovt, Karnataka for funding support. Abstract: Eragrostiella bifaria (EB) is a grass which belongs to Poaceae family and is found mainly in tropical regions of Asia, Africa, and Australia. It is found growing in rock crevices and is known to have desiccation tolerant traits. It can survive complete water loss and resurrect under minimal water conditions. Eragrostiella bifaria was collected in Devarayanadurga State Forest, Tumakuru district and identified based on its morphology. Field sampled Eragrostiella bifaria (EB) were subjected to physiological measurements on desiccation followed by quantification of various antioxidant enzymes. During desiccation relative water content (RWC) decreased to 10 % after 12 hrs of water loss and the plants showed intense inward curling. During rehydration, the RWC of the detached plants regained 92 % of its relative water content within 4 hrs. The rehydrated plants regained its original morphology. Further, there was an increase in activities of antioxidant enzymes namely superoxide dismutase, peroxidase, catalase and glutathione reductase, lipid peroxidation, and proline during desiccation. Also the plant showed variable responses to sucrose and starch content during desiccation. This physiological study revealed that Eragrostiella bifaria is metabolically potent to tolerate desiccation. Keywords: Desiccation stress, Eragrostiella bifaria, Proline, Reactive Oxygen Species, Resurrection plants. 1. Introduction Water deficiency is the most common abiotic stress factor for land plants. Extreme Loss of water or desiccation (10 % relative water content and below) is tolerated only by seeds, some pollen grains, and by a small group of specialized resurrection plants. Desiccation-tolerance is defined as the ability of losing water to air-dryness and returning to normal function when water is available (Gaff, 1971). Majority of plants are able to produce desiccation-tolerant seeds or spores, but the ability of tolerating desiccation in vegetative tissues is restricted to few species (Bernacchia and Furini, 2004). Therefore only a small portion of vascular plants and non-vascular plants are able to tolerate extreme desiccation and return to their normal metabolic function after rehydration. These plants possess physiological mechanisms and morphological structures that enable them to protect themselves against damage caused by extreme desiccation (Scott, 2000). Desiccation tolerance (DT) developed early in the evolution of the land plants and has been believed to be essential for the transition to dry land from fresh water (Oliver et al. 2000). DT requires the re-organization of physiological mechanisms in seeds/vegetative tissues which will enable plants to colonize habitats with less or no humidity. DT plants are classified into poikilochlorophyllous desiccation tolerant (PDT) and homoiochlorophyllous desiccation Volume XI, Issue XII, December/2019 Page No:639 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367 tolerant (HDT).During desiccation HDT species retain their chlorophylls and photosynthetic apparatus in readily recoverable state for e.g. Craterostigma spp. retain the thylakoid and chlorophyll membranes intact during desiccation. Approximately 300 angiosperm plant species can be considered to be resurrection type and can be found across the globe. Resurrection grasses are mostly found in arid and semi-arid areas of tropical and subtropical regions of the world, particularly in Africa and Australia (Porembski and Barthlott, 2000; Rascio and La Rocca, 2005; Toldi et al. 2009). However, they can also be found in some humid forests such as the Western Ghats of India. Desiccation tolerance in angiosperms has been mainly studied in the dicotyledonous plant Craterostigmaplantagineaum (Rodriguez et al., 2010; Petersen et al;, 2012; Gasulla et al., 2013), Craterostigmawilmsii (Cooper and Farrant 2002; Vicré et al., 2004a, b) and Myrothamnusflabeliflolius (Moore et al., 2005; 2006; 2007) and the monocotyledonous Xerophytavesiculosa (Collett et al., 2003; Peters et al., 2007; Ingle et al., 2008; Beckett et al., 2012). Grasses are among the world’s most agriculturally and economically valuable plant species (e.g. wheat, corn, rice etc.), having being selected over thousands of years of human development for crops and forages. Unlocking or developing grass crops and forages which are capable of vegetative DT would provide a significant economic and agricultural advantage to countries and regions prone to acute and changing periods of water stress. To date there is only little knowledge on desiccation tolerance in the grasses especially poaceae (Van der Willigen 2001; 2003; Basalmo 2005). Understanding desiccation tolerance in poaceae will enable to improve resistance to water stress in most economically important food crops. Since several major crops are monocots and employ C4 photosynthesis, understanding how resurrection monocots like EB respond to the dehydration of their vegetative tissues is critically important for better management of crops which fall extreme water stress. Therefore in this study we test the physiological potentials of Erogrostellia bifaria during desiccation and its responses tradeoff. 2. Materials and Methods: 2.1 Collection and identification of samples Selection of site for sample collection was conducted based on the Literature available in various databases on desiccation tolerance plants, availability and access for the samples, Knowledge of plants and their local ecology. Based on the above criteria, Extensive field survey was undertaken to collect grass samples from Devarayanadurga state forest of Tumakuru district, Karnataka. (Coordinates: 13.3737° N, 77.2075° E) during monsoon and non-monsoon season. The samples were collected and placed in polythene bags, labelled, transported in ice box to the laboratory. The collected samples were identified based on morphological characters such as a) morphology of leaves, b) rhizome and c) spikelets. 2.2 Relative Water Content Analysis: Plants of homogeneous age were selected whose aerial parts were similar in size and belonged to the same habitat. Field collected healthy and youngErogrostellia bifaria plants were allowed to hydrate in a petri dish (150mm X 20mm Size) flooded with double distilled water, until it did not further gain weight or till it weight saturation. Such types of plant tissue were considered as hydrated (H). The hydrated tissues were allowed to desiccate in room temperature until no more weight loss occurred and reached saturation such tissues were considered as desiccated Volume XI, Issue XII, December/2019 Page No:640 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367 (D). The desiccated tissues were further rehydrated under similar conditions until no further weight gain took place. These plant tissues were considered as rehydrated (R). All the plant materials required for further analysis were treated similarly. Then hydrated desiccated and rehydrated tissues were ground using liquid nitrogen and stored in - 80°C for the further analysis. The water content in the samples was calculated as the difference between fresh weight and dry weight divided by the fresh weight [Pandey 2010]. RWC was expressed in percentage. 2.3 Chlorophyll Measurements 0.5 g of hydrated, desiccated and rehydrated Eragrostiella bifaria leaf tissues were frozen using liquid nitrogen and homogenized using mortar and pestle. From the homogenized samples, chlorophyll was extracted using 10 mL 80% acetone. The test tubes were wrapped with aluminum foil and left at room temperature overnight, then crude extract was centrifuged at 3000g for 5 min using Centrifuge 5400R (Eppendorf CA USA) and the supernatant was collected while the pellet was discarded. The collected supernatant was read at 663.6 nm, 646.6 nm and 440.5 nm by using Bio Spectrometer Kinetic (Eppendrof CA USA), these are the major absorption peaks of chlorophylls a, b, and carotenoids, respectively [Poraet al 1989]. The total chlorophyll (Chla+b) contents were calculated using extinction coefficients provided by Poraet al 1989. - 2.4 Quantification of Superoxide Radical (O2 ) Super oxide was analysed according to (Fontana 2001). 1g of H, D and R states of Eragrostiella bifaria were extracted with 100mM potassium phosphate buffer pH 7.2 (2ml). To the reaction mixture 500 µl of 2mM nitro blue tetrazolium (NBT) was added and the incubation was continued for 20 more min. the reaction was stopped by the addition of 2ml 1.4-dioxan. The tubes were placed in water bath at 70°C for 15 minutes, cooled, centrifuged at 2000rpm using for 10 minutes to allow the cells to settle and the absorbance of the supernatant was measured at 540nm. Quantity of super oxide radical was expressed in µmol/g FW. 2.5 Estimation of Lipid Peroxidation Lipid peroxidation of all the Hydrated, desiccated and rehydrated plants of Eragrostiella bifaria were determined, as 2-thiobarbituric acid (TBA) reactive
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