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 family and is found mainly in tropical regions of , , and . 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 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 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

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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 . 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

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(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 metabolites chiefly malondialdehyde (MDA) as described by [Li 2000]. 0.2g of the tissues was extracted in 2 ml of 0.25% TBA made in 10% TCA. The reaction mixture was heated at 95°c for 30 minutes then cooled quickly. Then the samples were centrifuged at 10,000g for 10 minutes and absorbance of the supernatant was read at 532nm and 600nm, by subtracting the absorbance value taken at 600 nm, correction of non-specific turbidity was carried out. The lipid peroxidation level was expressed in n mol g-1 FWof MDA made using an extinction coefficient of 155 mM cm-1. 2.6 Analysis of Antioxidant Enzymes Four antioxidant enzymes (SOD, CAT, POD, and GR) were analyzed in H, D and R tissues ofErogrostellia bifaria. Tissues were ground separately in 6 mL of extraction buffer-1 (50 mM PBS, pH 7.8 for SOD and CAT assays) and 6 mL extraction buffer-2 (100 mM PBS, pH 7.0 for POD and GR assays) at 4 °C. The homogenates were collected and centrifuged at 15000 g at 4 °C for 20 min. SOD was assayed on the basis of its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT). A 6 mL reaction mixture of SOD consists of 50 mM PBS (pH 7.8), 130 mM methionine, 750 µM nitro blue tetrazolium chloride (NBT), 100 µM EDTA-Na2+, 20 µM riboflavin, and 0.1 mL of enzyme extract. The reaction solution was incubated for 10 min under fluorescent light

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with an intensity of 50 µmol m-2 s-1 for 20 min. The absorbance was determined at 560 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit photochemical reduction of NBT by 50% and -1 expressed in Unitsmg protein. CAT activity was assayed by measuring the initial rate of disappearance of H2O2 by the technique described by [Change and Maethly 1995]. The decline in absorbance at A240 was recorded by

BioSpectrometer (Eppendorf CA USA), and the activity was expressed in µmol of H2O2 catalyzed by a unit of CAT per min and were expressed in units/mg of protein. POD activity was analyzed using a modified method [Raoet al.

1995]. The reaction solution contained 100 mM PBS (pH 7.0), 50 mM o-methoxyphenol, 40 mM H2O2, and 0.1 mL of enzyme extract and was expressed in µmolmin-1g-1 protein. GR activity was determined by the method described

by [Halliwell and Foyer 1978]. The reaction solution consisted of 50 mMTris-HCl, 0.5 mM GSSG, 5 mM MgCl2, and 0.2 mM NADPH. GR activity was observed at 340 nm, within 3 min and expressed as the number of µmol of NADPH oxidization and was expressed in Units mg-1 protein. 2.7 Determination of Soluble Sugar and Starch The frozen hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria were ground to fine powder in a ° mortar with ice-cold 1M perchloric Acid (HClO4), then the extract was centrifuged at 12 000 g for 2 min at 4-8 C.

The supernatant was neutralized with 5M Potassium carbonate (K2CO3) and precipitated potassium perchlorate

(KClO4) was removed by centrifugation (Eppendorf CA USA). The supernatant was kept on ice and used for the estimation of sucrose, while the pellet was used for the determination of starch. Sucrose and starch were estimated enzymatically according to the Jones method [Pandey 2010]. Sucrose and starch content was expressed as µmol/g FW. 2.8 Proline Estimation Free proline accumulation of EB in H, D and R tissus were determined using the method of [Li et al 2000] 0.04 g of EB leaves were homogenized with 3% sulfosalicylic acid and after 72 hours proline released was measured. The homogenate was centrifuged at 3000 g for 20 minutes. The supernatant was treated with acetic acid ninhydrin, let to boil for 1 hour and then absorbance at 520 nm was determined. Contents of proline were expressed as mg g-1 dw-1. 2.9 Statistical analysis Data obtained was subjected to a one-way analysis of variance (ANOVA). Significant differences among the test groups were (P≤0.05) obtained by Tukey’s honestly significant difference (HSD), post hoc test using SPSS software (SPSS20.0, SPSS Inc., USA) Values shown in the Figures are the means ± standard errors (SEs) of three independent replicates. 3. Results

3.1Eragrostiella bifaria Morphological Identification Eragrostiella bifaria was identified based on the following morphological features. This grass was found to be green small structural perennial herb. Rhizome of Eragrostiella bifaria contained tufted culms, growing up to 80cm long. The leaves Blades were linear, involute, and sparsely hairy. The Spikelets were straw coloured, laterally compressed, linear to ovate oblong.

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3.2 Relative Water Content: Relative water content (RWC) is the technique for measuring of plant water balance status in terms of cellular water deficit due to desiccation.RWC of Eragrostiella bifaria was estimated in three independent experiments using whole plants under hydrated, desiccated and rehydrated conditions invitro. At each stage, photos of plants were captured with Nikon digital camera D3200 (USA). During desiccation the plants maintained their RWC for first few hours before losing 80% water. Plants reached an air dry state i.e. 10% RWC after 12 hours. Upon rewatering the water level gradually rose to around 92% RWC.

Fig 01: various stages of water loss and gain in Eragrostiella bifaria A-Hydrated, B-Desiccated and C-Rehydrated.

3.3 Changes in chlorophyll content due to desiccation stress Decrease in chlorophyll content of leaves is thought to be linked to the protection of plants against UV light and from damage as a result, oxygen free radical generation during desiccation (Sherwin and Farrant,1998). Illustrated in figure 02 is the total chlorophyll content (chla+chl b) in hydrated, desiccated and rehydrated plants of Eragrostiella bifaria. We observed that there were significant differences in the chla+chl b among Hydrated, desiccated, rehydrated states of Eragrostiella bifaria. The chla+chl b in desiccated decreased to 6.54+1.89µg g-1 from 10.42+3.54µg g-1. This shows that approximately 60 % of the total chlorophyll of the hydrated tissues was retained during the desiccation. However, during rehydration chlorophyll content was found to be 9.55+4.27µgg-1. This shows that chloroplasts recovered and regain its normal function during rehydration. In Eragrostiella bifaria chlorophyll content of the leaves did not drop too much in desiccated state indicating that no complete dismantling of photosynthetic apparatus took place and shows a mechanism of protecting chlorophyll content from total destruction during desiccation.

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15 B A A 10

fw) 5

0

Chlorophyll a and g (µg/ and a b Chlorophyll Hydrated Desicated Rehydrated CHL a CHL b

Fig 02: Relative water content in percentage (A) and total chlorophyll (chla+chl b) in µg/g of fresh weight (B) in hydrated, desiccated and rehydrated states of Eragrostiella bifaria. 3.4 Activity of Superoxide Radical due to desiccation Superoxide concentrations were expressed as nmol/gm fresh weight and were quantified in hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria. There was significant Increase in superoxide concentration during the desiccation stage as it increased from 0.22 nmol/gm in hydrated stage to 0.67 nmol/gm. During the rehydration stage it was found to be 0.24 nmol/gm FW. There was a significant change in percentage of superoxide radical level in desiccated state. It increased 20 times when compared to hydrated state and decreased during rehydration. Increased rate of superoxide radical level in desiccated state of Eragrostiella bifaria clearly suggest that there was an oxidative stress due to desiccation. 3.5 Lipid Peroxidation: The main cellular components susceptible to damage by free radicals are lipids (peroxidation of unsaturated fatty acids in membranes), lipid peroxidation indicates oxidative stress in response to desiccation which will result in damage to membranes and inactivation of enzymes, i.e., resulting in loss of cell viability (Mittler R, (2002); Mattos LM, and Moretti CL 2015). Lipid peroxidation in Eragrostiella bifaria was estimated as reactive metabolites of 2- TBA mainly MDA. In hydrated state it is found to be 0.443 nmol g-1 FW. In desiccated state MDA was found to be 0.403 nmol g-1 FW. Further it is increased to 0.429 nmolg-1 FW in rehydrated state of Eragrostiella bifaria (EB). The percentage difference between hydrated and desiccated state is around 10%, there is slight increase in percentage between desiccated to rehydrated i.e around 7% and when compare to hydrated and rehydrated the percentage difference is around 3%. The decrease in lipid peroxidation in desiccated state indicates protection against oxidative damage and membrane integrity.

0.8 0.6 C D 0.6 ) 0.4 0.4

nmol/g 0.2

0.2 (

(nmol/gm) (nmol/gm) FW lipid peroxidationlipid Superoxideradical 0 0 H D R H D R Fig 03: concentration of superoxide radical (C) and MDA (D) in hydrated, desiccated and rehydrated states of Eragrostiella bifaria due to desiccation stress.

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3.6 Activity of antioxidant enzymes in response to desiccation In order to eliminate the excessive oxidative stress, the antioxidant mechanisms are generated. In the plant there was a significant change in the activities of four Antioxidant enzymes, superoxide dismutase (SOD), Peroxidase (POD), Catalase (CAT) and Glutathione reductase (GR), during desiccation. The activities of all four enzymes increased gradually during desiccation which returned to approximately original levels during Rehydration. The concentration of SOD in Eragrostiella bifaria (EB) increased from 10.617 unitsmg-1 protein in hydrated to 18.38 unitsmg-1proteinin desiccated stage and again it decreased to13.65 unitsmg-1protein upon rehydration. The •− SOD activity is responsible for the scavenging of the O2 . The percentage increase in desiccated was 54% and in rehydrated was 31% when compared to hydrated stage. The concentration of catalase also increased to 23.54 units/mg of protein in desiccated stage from 18.26 units/mg of

protein in hydrated and which reduced to 19.98 units/mg of protein in rehydrated stage. Excessive level of H2O2 is minimized by the activity of catalase. When compared to hydrated the percentage of increase in desiccated is 25% and in rehydrated is 9%. Similarly, the peroxidase concentration also increased in desiccated, when compare to hydrated and rehydrated stage i.e. 660.754 units/mg of protein, 375.554 units/mg of protein, and 382.254 units/mg of protein respectively. An increase of 55% in desiccated when compared to control (hydrated). The Glutathione reductase increased in desiccated i.e. 353.3 units/mg of protein from 229.7 units/mg of protein in hydrated which accounts an increase of 42% and 217.4 units/mg of protein in rehydrated stage. From the obtained results it clearly shows all antioxidant enzymes increased in desiccated stage compare to hydrated and little minimized in rehydrated. It indicates that during the desiccation stress, antioxidants are overexpressed to eliminate the oxidative stress.

E F

G H

Fig 04: Specific activity of (E) Superoxide dismutase, (F) Peroxidase, (G) catalase and (H) Glutathione reductase in hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria.

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3.7 Soluble Sugars and Starch content due to desiccation stress A range of non-reducing oligosaccharides (e.g. sucrose, sorbitol, trehalose and the raffinose family of oligosaccharides) accumulate in resurrection plants mainly through breakdown of starch during dehydration (Crowe et al., 1998; Mundree et al., 2000; Crowe et al., 2001; Peters et al., 2007; ElSayed et al., 2014). Among all non-reducing oligosaccharides, sucrose is the main product (Bianchi et al., 1991; Proctor and Tuba, 2002; Nar et al., 2009) and the predominant carbohydrate involved in mechanisms of protection (Scott, 2000). Sucrose content was expressed as µmol/g FW and it showed a decrease in the hydrated and rehydrated tissues of Eragrostiella bifaria. The sucrose concentration was found to be 0.443, 0.664 and 0.429 µmol/g FW in hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria respectively i.e. around 40% increase in desiccated and 3% difference in rehydrated stage when compared to hydrated stage. The concentrations of soluble sugars increased where as there is decrease in starch concentration in desiccated stage. The starch concentration was found to be 0.435, 0.203 and 0.418 µmol/g FW in hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria respectively which accounts an 73% in desiccated and 4% in rehydrated when compared to control ( hydrated). It implies that increased fraction of sucrose was go together with decrease in starch fraction in desiccated state of Eragrostiella bifaria. Sugars protect the cells during desiccation by maintaining hydrophilic interactions in proteins and membranes by substituting water from Hydroxyl groups of sugars. Sugars are considered are a key contributing factor to vitrification.

I J 0.6 0.8 0.7 0.5 0.6 0.4 0.5

0.3 0.4 µmol/g FW) µmol/g µmol/g FW) µmol/g 0.3 0.2 0.2 0.1

Starch( 0.1 sucrose( 0 0 H D R H D R Fig 05: levels of (I) Starch and (J) Sucrose in hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria. 3.8 Changes in Proline Accumulation due to desiccation stress Proline is a low-molecular-weight osmolyte which accumulates during water deficit in plants. Proline levels in Eragrostiella bifaria was expressed in terms of µmol/g FW. It was clearly evident that the proline levels in desiccated state increased rapidly. In Hydrated stage it was found to be 0.012 µmol/g FW and desiccated stage proline content increased to 0.035µmol/g FW. During rehydration it was found to be 0.014 µ mol/g FW. Which corresponds to 98% in desiccated and 15% in rehydrated when compared to control (hydrated stage). These results show that drastic increase in the proline concentration during desiccation indicates the role of proline in cellular protection of DT plants. Proline functions like an osmolyte, a scavenger of ROS, and also a molecular chaperone.

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0.05 K 0.04

0.03

0.02

0.01 proline(µmol/gFW) 0 H D R Fig 06: concentrations of proline accumulation in (K) in hydrated, desiccated and rehydrated tissues of Eragrostiella bifaria. 4. Discussion RWC is theappropriate technique for measure the plant water balance status in terms of cellular water deficit associated with physiological consequences. Water deficit induces a number of structural changes in plants in order to increase water use efficiency. The most obvious of which is leaf folding (Le and McQueen-Mason, 2006; Nar et al., 2009). In this study of Eragrostiella bifaria showsdecreased RWC in desiccated stage i.e. 10 % and during rehydration, the RWC of the desiccated sample regained up to 92% and the rehydrated sample regained broadly the original morphology. Resurrection plants respond to dehydration faster and reduce photosynthesis relatively early from 80 % RWC and below (Farrant, 2000). If dehydration continues, resurrection plants terminate photosynthesis. In poikilochlorophyllous resurrection plants, chlorophyll pigments are disintegrated and thylakoid membranes are dismantled to prevent the production of ROS through photosynthesis. Homoiochlorophyllous resurrection plants retain much of their photosynthetic apparatus but limit photosynthesis through shading, pigmentation and/or reduction of leaf surface area (Hallam and Luff, 1980; Gaff, 1981; Sherwin and Farrant, 1998; Tuba et al., 1998; Farrant, 2000; Farrant et al., 2003).

Plants display a variety of physiological responses at the cellular and whole-organism levels during dehydration and rehydration. Some of the key changes include photosynthesis, stomatal closure, pigmentation and respiration. These changes are mainly to prevent oxidative damage resulting from ROS accumulation during dehydration. One of the main problems plants face during dehydration is oxidative stress resulting from the accumulation of reactive oxygen species (ROS). This ROS accumulation is mainly due to the disruption of the photosynthesis electron chain during dehydration. During dehydration resurrection plants produce high amount of antioxidants, meanwhile in order to retain their systems for rehydration [Kranner et al. 2002]. Desiccation process can damage lipid membranes and proteins by producing a number of reactive oxygen species (ROS). By using effective mechanism S. stapfianus stimulate free radical scavenging enzymes for example ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase to eliminate the ROS [Sgherri et al. 1994]. Desiccation enhances the antioxidant activity in other resurrection plants also [Sgherri et al. 1994; Sherwin and Farrant 1998].

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Glutathione reductase (GR), ascorbate peroxidase (APX) and superoxide dismutase (SOD) are antioxidant enzyme which shows good response to several abiotic stresses in both desiccation sensitive as well as desiccation tolerant organisms, and are known as common ‘housekeeping’ protectants [Illing et al. 2005]. In Desiccation tolerant tissues of these enzymes stay elevated at the time of dehydration. This may be a significance of mechanisms that defend and sustain the antioxidant enzymes in their natural states, which results in improved activity of the proteins, relatively unique desiccation tolerance mechanism (Illing et al. 2005).

In Eragrostiella bifaria, there was significant change in the activities of four Antioxidant enzymes, superoxide dismutase (SOD), Peroxidase (POD), Catalase (CAT) and Glutathione reductase (GR). All antioxidant enzymes increased in desiccated compare to hydrate and little minimized in rehydrated. It indicates that during the desiccation stress antioxidants are overexpressed to overcome the oxidative stress. Antioxidant activity might be particularly critical for homoiochlorophyllous resurrection plants as the maintained chlorophyll could be a source of ROS production during desiccation. Kranner et al. (2002) demonstrated an increased accumulation of antioxidants in desiccated M. fabellifolia. This study also showed the amount of broken down antioxidant in the desiccated tissue has a direct relation with the period of desiccation. The accumulation of sugars during drying is an integral part of vegetative (as well as propagative) desiccation tolerance. Sugars play an important role in Osmotic Adjustment in grasses (Homayouniand Khazarian, 2014). Sucrose, the principal osmoprotectant in many species, is synthesized from carbon originating from reserve sugars such as starch in most resurrection plants or from octulose in C. plantagineum (Omarova, Bogdanova and Polimbetova,1995). Desiccation of Craterostigmaalso induces a major change in carbohydrate metabolism during water loss that may be directly related to desiccation tolerance. Such increases in sucrose and other sugars or sugar derivatives occur in several desiccation-tolerant species: e.g., sucrose and trehalose in MyrothamnusflabellifoliaWelw. (Bianchi et al. 1993; Drennan et al. 1993), sucrose in Boeahygroscopica(F.) Proline content is another component of osmotic regulation in plants. Plants species and duration of the stress were found to affect proline contents. Proline is a low-molecular-weight osmolytes which accumulates during water deficit in plants. Accumulation of proline is involved with the osmotic adjustment in the cell (Wahid and Close, 2007). Proline is also involved with the vitrification state and protection of the membrane and other macromolecules in desiccation tolerant plants. The increase in proline concentration was also shown to occur in the resurrection grass E. nindensis(Vander Willigenet al.2004). Conclusion Desiccation induced several changes in the Eragrostiella bifaria. The results obtained from this study suggest that desiccated Eragrostiella bifaria have the ability to recover complete physiological activities following rehydration, as all the measured traits returned to the control level. It is proposed that the tolerance to desiccation in vascular resurrection plants is due to the combinatorial effect of accumulation of high levels of antioxidants together with various osmoprotective compounds such as sugars, in particular sucrose and proline.

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Reference:

Balsamo RA, Vander Willigen C, Boyko W, Farrant J. 2005. Anomalous leaf tensile properties during dehydration may help elucidate mechanisms of desiccation tolerance in Eragrostisnindensis. PhysiologiaPlantarum124: 336– 342.

Beckett, M.; Loreto, F.; Velikova, V.; Brunetti, C.; DiFerdinando, M.; Tattini, M.; Calfapietra, C.; Farrant, J.M. (2012) Photosynthetic limitations and volatile and non-volatile isoprenoids in the poikilochlorophyllous resurrectionplant Xerophytahumilis during dehydration and rehydration. Plant Cell Environ.35, 2061–2074.

Bernacchia, G., and Furini, A. (2004). Biochemical and molecular responses to water stress in resurrection plants. PhysiologiaPlantarum121, 175-181. doi: 10.1111/j.1399-3054.2004.00321.x.

Bianchi, G., Gamba, A., Murelli, C., Salamini, F., and Bartels, D. (1991).Novel carbohydrate metabolism in the resurrection plant craterostigmaplantagineum.The Plant Journal1, 355-359.

Bianchi G, Gamba A, Limiroli R, Pozzi N, ElsterRl, Salamini F, Bartels D (1993) The unusual sugar composition in leaves of the resurrection plant Myrothamnus ¯abellifolia. PhysiologiaPlantarum87, 223-226.

Change B, Maehly AC (1995) Assay of catalases and peroxidase. Meth Enzymol2, 764-775. Collett H. M., Butowt R., Smith J., Farrant J., Illing N. (2003). Photosynthetic genes are differentially transcribed during the dehydration-rehydration cycle in the resurrection plant, Xerophytahumilis.J. Exp. Bot.54, 2543–2595 10.1093/jxb/erg285

Cooper K., Farrant J. M. (2002). Recovery of the resurrection plant Craterostigmawilmsii from desiccation: protection versus repair.J. Exp. Bot.53, 1805–1813 10.1093/jxb/erf028

Crowe, J. H., Crowe, L. M., Carpenter, J. F., Rudolph, A. S., Aurell-Winstrom, C., Spargo, B. J. &Anchordoguy, T. J. (1988). Interactions of sugars and membranes.Biochim.Biophys.Acta947: 367–384.

Crowe, J.H., Crowe, L.M., Oliver, A.E., Tsvetkova, N., Wolkers, W., and Tablin, F. (2001). The trehalose myth revisited: Introduction to a symposium on stabilization of cells in the dry state. Cryobiology 43, 89-105. doi: http://dx.doi.org/10.1006/cryo.2001.2353.

Drennan, P., Smith, M., Goldsworthy, D., and Van Staden, J. (1993).The occurrence of trehalose in the leaves of the desiccation-tolerant angiosperm myrothamnusflabellifoliuswelw.Journal of plant physiology 142, 493-496.

Elsayed, A., Rafudeen, M., and Golldack, D. (2014). Physiological aspects of raffinose family oligosaccharides in plants: Protection against abiotic stress. Plant Biology 16, 1-8.

Farrant, J.M., Vander Willigen, C., Loffell, D.A., Bartsch, S., and Whittaker, A. (2003).An investigation into the role of light during desiccation of three angiosperm resurrection plants.Plant, Cell & Environment 26, 1275-1286.

J.M. Farrant. (2000) A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species, Plant Ecol. 151, 29–39.

Fontana M, Mosca L, Rosei MA (2001) Interaction of enkephalins with oxyradicals, Biochemical Pharmacology61, 1253-1257.

Gaff DF (1971) Desiccation-tolerant flowering plants in southern africa. Science 174, 1033-1034.

Volume XI, Issue XII, December/2019 Page No:649 The International journal of analytical and experimental modal analysis ISSN NO: 0886-9367

Gaff DF (1981) The biology of resurrection plants. Melbourne: University of Western Australia Press.

Gasulla F, VomDorp K, Dombrink I, Zahringer U, Gisch N, Dormann P (2013). The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigmaplantagineum: a comparative approach. Plant J. 75, 726–741.

Hallam, N.D., and Luff, S.E. (1980).Fine structural changes in the mesophyll tissue of the leaves of xerophytavillosaduring desiccation.Botanical Gazette 141, 173-179. doi: 10.2307/2474849.

Halliwell B, Foyer CH (1978) Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography. Planta139, 9-17.

Homayouni, H.andV.Khazarian, 2014. Effect of deficit irrigation on soluble sugars, starch and proline in three corn hybrid.Indian. J. Sci.Res.,7(1): 910-917

N. Illing, K.J. Denby, H. Collett, A. Shen, J.M. Farrant. (2005) The signature of seeds in resurrection plants: a molecular and physiological comparison of desiccation tolerance in seeds and vegetative tissues, Integr. Comp. Biol.45, 771–787.

Ingle RA, Collett H, Cooper K, Takahashi Y, Farrant JM, IllingN(2008) Chloroplast biogenesis during rehydration of the resurrection plant Xerophytahumilis: parallels to the etioplastchloroplasttransition. Plant Cell Environ31, 1813–1824.

Kranner, I., Beckett, R.P., Wornik, S., Zorn, M., and Pfeifhofer, H.W. (2002). Revival of a resurrection plant correlates with its antioxidant status. The Plant Journal 31, 13-24.

Le T. N., McQueen-Mason S. J. (2006). Desiccation-tolerant plants in dry environments. Rev. Environ Sci. Biotechnol.5, 269–279. 10.1007/s11157-006-0015-y

Li HS, Sun Q, Zhao SJ, Zhang WH, Ed. (2000) In Principles and Techniques of Plant Physiological Biochemical Exp.

Mattos LM &Moretti CL. (2015) Oxidative Stress in Plants under Drought Conditions and the Role of Different Enzymes.Enzyme Engineering5,136.

Mittler R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science7,405-410

Moore J. P., Westall K., Ravenscroft N., Farrant J., Lindsey G., Brandt W. (2005). The predominant polyphenol in the leaves of the resurrection plant Myrothamnusflabellifolius, 3,4,5 tri-O-galloylquinic acid, protects membranes against desiccation and free radical-induced oxidation.Biochem. J.385, 301–308 10.1042/BJ20040499

Moore, J.P., Cannesan, M.A., Chevalier, L.M., Lindsey, G.G., Brandt,W., Lerouge, P., Farrant, J.M. and Driouich, A. (2006) Theresponse of the leaf cell wall to desiccation in the resurrection plant Myrothamnusflabellifolius. Plant Physiol. 141, 651–662

Moore JP, Hearshaw M, Ravenscroft N, Lindsey GG, Farrant JM, Brandt WF (2007) Desiccation-induced ultra structural and biochemical changes in the leaves of the resurrection plant myrothamnusflabellifolia. Australian Journal of Botany 55, 482-491.

Mundree, S.G., Whittaker, A., Thomson, J.A., and Farrant, J.M. (2000).An aldose reductase homolog from the resurrection plant xerophytaviscosabaker.Planta211, 693-700.;

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Nar, H., Saglam, A., Terzi, R., Várkonyi, Z., and Kadioglu, A. (2009). Leaf rolling and photosystem ii efficiency in <i>ctenanthesetosa</i> exposed to drought stress. Photosynthetica47, 429-436. doi: 10.1007/s11099- 009-0066-8.

Omarova, E.I., Bogdanova, E.D., and Polimbetova, F.A. (1995).Regulation of water-loss by the leaves soft winter- wheat with different organization of leaf structure.Russian Journal of Plant Physiology 42, 383-385.

Oliver MJ, Tuba Z, Mishler BD (2000) Phylogeny of desiccation-tolerance in land plants.Plant Ecol. 151(1) in this issue.

Pandey, S. Ranjan, F. Deeba, A.K. Pandey, R. Singh, P.A. Shirke, U.V. Pathre. (2010) Desiccation-induced physiological and biochemical changesin resurrection plant, Selaginellabryopteris, J. Plant Physiol. 167, 1351–1359

Peters, S., Mundree, S.G., Thomson, J.A., Farrant, J.M., and Keller, F. (2007). Protection mechanisms in the resurrection plant xerophytaviscosa(baker): 150 References Both sucrose and raffinose family oligosaccharides (rfos) accumulate in leaves in response to water deficit. Journal of experimental botany 58, 1947-1956

Petersen J., Eriksson S. K., Harryson P., Pierog S., Colby T., Bartels D., et al. (2012). The lysine-rich motif of intrinsically disordered stress protein CDeT11-24 from Craterostigmaplantagineum is responsible for phosphatidic acid binding and protection of enzymes from damaging effects caused by desiccation.J. Exp. Bot.63, 4919–4929 10.1093/jxb/ers173

Pora RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: Verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. BiochimicaetBiophysicaActa (BBA)-Bioenergetics 975, 384-394.

Porembski S, Barthlott W (2000) Granitic and gneissic outcrops (inselbergs) as centers of diversity for desiccation- tolerant vascular plants. Plant Ecology 151,19-28.

Proctor MCF &Tuba Z (2002) Poikilohydry and homoihydry: Antithesis or spectrum of possibilities? New Phytologist156, 327-349

Rascio N, La Rocca N (2005) Resurrection plants: The puzzle of surviving extreme vegetative desiccation. Critical Reviews in Plant Sciences 24, 209-225.

Rodriguez, M.C.S., Edsgärd, D., Hussain, S.S., Alquezar, D., Rasmussen, M., Gilbert, T., Nielsen, B.H., Bartels, D., and Mundy, J. (2010).Transcriptomes of the desiccation-tolerant resurrection plant craterostigmaplantagineum. The Plant Journal 63, 212-228. doi: 10.1111/j.1365-13X.2010.04243.x.

Scott P. (2000) Resurrection plants and the secrets of eternal leaf.Annals of Botany.85, 159–166

Sgherri, C.L.M., Logginin, B., Bochicchio, A. and Navari-Izzo, F.(1994) Antioxidant system in Boeahygroscropica: changes inresponse to desiccation and rehydration.Phytochem, 73, 277–381.

Sherwin H, Farrant J (1998) Protection mechanisms against excess light in the resurrection plants craterostigmawilmsii and xerophytaviscosa. Plant Growth Regulation 24, 203-210.

Toldi O, Tuba Z, Scott P (2009) Vegetative desiccation tolerance: Is it a goldmine for bioengineering crops? Plant Science176, 187-199.

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Tuba Z, Protor CF, Csintalan Z (1998) Ecophysiological responses of homoiochlorophyllous and poikilochlorophyllous desiccation tolerant plants: A comparison and an ecological perspective. Plant Growth Regulation 24, 211-217.

Vander Willigen C, Pammenter NW, Jaffer MA, Mundree SG, Farrant JM (2003) anultra-structural study using anhydrous fixation of eragrostisnindensisa resurrection grass with both desiccation-tolerant and -sensitive tissues. Functional Plant Biology 30, 281-290.

Vander Willigen C, Pammenter NW, Mundree SG, Farrant JM. (2001). Some physiological comparisons between the resurrection grass, Eragrostisnindensis, and the related desiccation-sensitive species, E. curvula. Plant Growth Regulation35: 121–129.

Vander Willigen, C., Pammenter, N.W., Mundree, S.G., and Farrant, J.M. (2004). Mechanical stabilization of desiccated vegetative tissues of the resurrection grass eragrostisnindensis: Does a tip 3;1 and/or compartmentalization of subcellular components and metabolites play a role? Journal of experimental botany 55, 651-661.doi: 10.1093/jxb/erh089

Vicré M, Farrant JM, Driouich A (2004a) Insights into the cellular mechanisms of desiccation tolerance among angiosperm resurrection plant species. Plant, Cell & Environment 27, 1329-1340.

Vicré M, Lerouxel O, Farrant J, Lerouge P, Driouich A (2004b) Composition and desiccation-induced alterations of the cell wall in the resurrection plant craterostigmawilmsii. Physiologia Plantarum120, 229-239.

Wahid A, Close TJ (2007) Expression of dehydrins under heat stress and their relationship with water relations of sugarcane leaves. BiologiaPlantarum51,104-109.

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