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Research Article
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Proteome Analysis of Detached Fronds from a Resurrection Plant Selaginella Bryopteris in Response to Dehydration and Rehydration
Farah Deeba1, Vivek Pandey* 1, Uday Pathre1, Sanjeev Kanojiya2
1Plant Physiology Lab, National Botanical Research Institute, Lucknow 226001, India
2SAIF, Central Drug Research Institute, Lucknow 226001, India
*Corresponding author: Vivek Pandey, Plant Physiology Lab, National Botanical Research Institute,
Lucknow 226001, India,
Fax    : +91-522-2205847,


Email : v.pandey@nbri.res.in
Received January 05, 2009; Accepted February 20, 2009; Published February 20, 2009
Citation: Farah D, Vivek P, Uday P, Sanjeev K (2009) Proteome Analysis of Detached Fronds from a Resurrection Plant Selaginella Bryopteris in Response to Dehydration and Rehydration. J Proteomics Bioinform 2: 108-116.
 
Copyright: ©2009 Farah D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
Abstract
Selaginella bryopteris (L.) Bak is a resurrection plant. Its detached fronds have unique ability to survive desiccation similar to that of whole plant. In order to understand the mechanisms of desiccation tolerance, proteome studies were carried out using fronds of the Selaginella bryopteris to reveal proteins that were differentially expressed in response to dehydration and rehydration. There was not much difference in electrolyte leakage between control, dehydrated and rehydrated fronds. During dehydration the plants showed only respiration and drop in Fv/Fm values. Both fluorescence and photosynthesis regained totally after rehydration. About 250 protein spots were reproducibly detected and analyzed. Analysis of the identi?ed proteins revealed that proteins involved in protein destination and degradation were more expressed in desiccated fronds. These findings tentatively indicate that some of the proteins could contribute a physiological advantage to S. bryopteris under desiccation.
Keywords:
Selaginella bryopteris; desiccation tolerance; fluorescence; two-dimensional electrophoresis

Introduction
Plants as sessile organisms have evolved a wide spectrum of adaptations to cope with the challenges of environmental stress. One major factor that limits the productive potential of higher plants is the availability of water. The International Water Management Institute predicts that by the year 2025, one-third of the world’s population will live in regions that will experience severe water scarcity (www.iwmi.org). Therefore, it has become imperative for plant biologists to understand the mechanisms by which plants can adapt to water deficit while retaining their capacity to serve as sources of food and other raw materials. Water deficit can affect plants in different ways. A mild water deficit leads to small changes in the water status of plants, and plants cope with such a situation by reducing water loss and/or by increasing water uptake (Bray, 1997). The most severe form of water deficit is desiccation—when most of the protoplasmic water is lost and only a very small amount of tightly bound water remains in the cell.

An important contribution to our understanding of the mechanism of desiccation tolerance is derived from ‘resurrection plants’, which can survive even with <5% of their total water in the vegetative tissues and are able to regain normal metabolism and growth within several hours of rewatering. (Ramanjulu and Bartels, 2002). Selaginella bryopteris (L.) Bak is one such resurrection plant. Another unique feature of S. bryopteris is the ability of detached fronds to possess a similar level of desiccation tolerance as that of whole plants. This was ascertained by doing various physiological parameters in intact plants as well as detached fronds (data not shown). The desiccation and rehydration of detached fronds avoid interference from developmental regulation and long-distance signalling from other organs (Jiang et al, 2007). We intent to use it as a model system to understand systems-level understanding of responses to desiccation using multiple platforms that provide information about global transcript levels, proteome, a wide range of metabolites, and enzyme activities, and growth parameters.

Proteins are responsible for maintaining all cellular functions and their production is governed by the genetic code. Stress responses in plants cause changes in the structure and activity of a protein. Therefore characterizing proteins and understanding their function is important for plant stress studies. Two-dimensional gel electrophoresis in conjunction with mass spectrometry is a powerful tool for identifying large number of proteins. These techniques, in combination with the constantly expanding genomic and EST databases, enable the simultaneous characterization/analysis of the expression profiles of a large set of proteins.

Here, we report a detailed study of the changes in protein expression that occur in dehydrated and rehydrated S. bryopteris detached fronds using two-dimensional polyacrylamide gel electrophoresis (2DE) and the identi?cation of 9 dehydration-responsive proteins by mass spectrometry. Analysis of the identi?ed proteins reveals that proteins involved in protein destination and degradation were more expressed in desiccated fronds. This finding indicate that these responses of S. bryopteris may be more of due to detachment of fronds rather than desiccation.

Material and Methods
Plants of S. bryopteris were collected from wild (Mirzapur district, U.P., India; latitude 23o52´ -25o32´ N & longitude 82o7´-83o33´ E) and maintained at Institutes’ fern house. Fronds with three different water statuses were used in the study: Control (C)-RWC 100%, dehydrated (S1)-RWC 10%, rehydrated (S2). Freshly detached fronds from well-hydrated plants were placed in Petri plates and subjected to dehydration in dark at 25 0C (60% relative humidity) in a growth chamber. Control samples of detached fronds were kept fully hydrated under the same condition. Rehydration was done by keeping the dehydrated fronds on wet filter paper for 12 h in dark.

Photosynthesis and chlorophyll fluorescence were recorded using LiCOR 6400 and PAM 2000, respectively.

Protein Extraction
Fronds were ground in liquid N2 and the resulting powder was extracted with 50 mM Tris-HCl, pH 8.0, 25 mM EDTA, 500 mM thiourea and 0.5% b-mercaptoethanol. The extract was mixed with 10% cold TCA and 0.07% BME, and left overnight at -20 OC. The mixture was centrifuged at 4500 rpm for 10 min and the pellet was washed three times with acetone and 0.07% BME. The pellet was then vacuum dried, solubilised in 0.1 M Tris-HCl, pH 8.0, 50 mM EDTA and 2% BME. Proteins were extracted with 2.5 ml Trisbuffered phenol and centrifuged at 4500 rpm for 10 min. After centrifugation, lower phenol phase was collected with the help of Pasteur pipette. To this 10 ml 0.1 M ammonium acetate in methanol was added and left overnight at -20 OC. The mixture was centrifuged at 4500 rpm for 10 min and pellet was dissolved in 0.1 M ammonium acetate in methanol and 1% BME. It was centrifuged at 6000 rpm for 10 min. It was washed twice with cold acetone. Pellet was dried and kept in -80 OC until further use. Total protein content was analyzed using the protein assay dye reagent (Bio-Rad).

Fifty µg protein was used for Isoelectric focussing (IEF) with 7 cm IPG strips, pH 4 to 7 in Ettan IPGphor unit. The IPG strips were rehydrated overnight with total protein diluted in 8 M urea, 2% CHAPS (w/v), 0.5% IPG buffer pH 4 to 7, 25 mM DTT, bromophenol blue up to a volume of 135 µl. After rehydration, focussing was done on Ettan IPGphor under following conditions: 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, and 2000 V for 4 h for a total of 10 kVh. Then strips were equilibrated in a buffer containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT for 15 min, and another 15 min in the same buffer but with 2.5% (w/v) iodoacetamide replacing DTT. The second dimension was run in Hoefer mini-gel apparatus in 7 x 8 cm homogeneous 12% SDS-PAGE gels. Electrophoresis was performed in a standard Tris-Glycine running buffer at a constant voltage of 200 V. Gels were silver stained and gel images were acquired with the BioRad Fluor-S Imager. The data was analyzed using ImageMaster 2D Platinum 5.0 software (Amersham Bioscience). Relative volume (% volume) was used to quantify and compare the spots. The criteria for defining the protein expression patterns were determined as follows: up-regulated, % volume increased at least twofold; down-regulated, % volume decreased at least two fold; unchanged, % volume varied within two-fold.

Protein Identification
Tryptic digestion of the protein spots excised from the gels, and sample preparation were performed according to Koistinen et al. (2002). Briefly, gel particles were destained and dehydrated by washing three times with 25 mM ammonium bicarbonate containing 50% acetonitrile. Destained particles were dried in a vacuum centrifuge concentrator and rehydrated in equal volumes of 0.1 µg µl-1 trypsin (Sigma) and 50 mM ammonium bicarbonate. Gel particles were immersed in 25 mM ammonium bicarbonate and samples were digested overnight at 37oC. Peptides were extracted twice with 50% ACN/5% TFA, gel particles were rehydrated with water, and two more extractions were performed. The recovered peptides were concentrated to a final volume of 20 µl.

The tryptic peptides were analysed using Thermo Finnigan LCQ Advantage max ion trap mass spectrometer having Finnigan Surveyor HPLC system connected to it. The column was Thermo Bio-Basic 100 X 1, 5 µM and solvent was eluted as given gradient program at 40 µl/min. The 2 µl sample was introduced into the ESI source through Finnigan Surveyor autosampler. The mass spectra were scanned in the range 300-1800 Da and the maximum ion injection time was set 50 nS. Ion spray voltage was set at 5.3 KV and capillary voltage 30.5 V. The MS scan run up to 20 min and the average of 2-6 scan at peak top in TIC were taken in to consideration. The MS/MS data were processed using BIOWORKS 3.1 SR1 and searched against NCBI nr protein sequence databases with the MS/MS ion searching program MASCOT (http://www.matrixscience.com). Ions score is –10 * log (P), where P is the probability that the observed match is a random event. Individual ions scores indicate identity or extensive homology, * indicates that P < 0.05; Protein scores are derived from ions scores as a nonprobabilistic basis for ranking protein hits.

Results and Discussion
Detached fronds from fully hydrated S. bryopteris were subjected dehydration and rehydration as described in material and methods. The RWC of detached fronds decreased rapidly from 100% (control) to a stable 10% after only 6 hrs. Dehydrated fronds showed intense inward curling (Fig 1). During rehydration, a RWC of 104% was achieved after 12 hrs and fronds regained broadly the original morphology. Leaf folding during drying of plants has been proposed to prevent light-chlorophyll interaction and thus light-induced damage (Farrant and Sherwin, 1998). Electrolyte leakage is used to test the integrity of cell during dehydration and rehydration. There was not much difference in electrolyte leakage between control, dehydrated and rehydrated fronds (Fig 2), indicating that S. bryopteris had a fundamental mechanism to survive desiccation. Farrant et al (1999) also reported similar findings with desiccation tolerant angiosperm Craterostigma wilmsii. S. bryopteris plant thus represents a simpli?ed system to investigate the basis of desiccation tolerance, taking advantage of avoidance of possible developmental regulation and long-distance signaling from other organs. The detached fronds in hydrated state showed Fv/ Fm values around 0.8 indicating the functional photosystems (Fig 3). After dehydration the plants showed net respiration and drop in Fv/Fm. Both fluorescence and photosynthesis regained totally after rehydration, further giving proof that detached, desiccated S. bryopteris fronds fully revived metabolism. In general, water de?cit causes a reduction in the photosynthesis rate, resulting in the decline in the photochemical ef?ciency of PSII and electron transport rate in desiccation-tolerant as well as desiccation-sensitive plants (Ekmekci et al. 2005). The decline in PSII activity could represent a protective mechanism from toxic oxygen production in order to maintain mem-brane integrity and to ensure protoplast survival (Di Blasi et al. 1998). However, only proteins within the thylakoid membranes of resurrection plants remain stable during desiccation and rehydration (Schneider et al., 1993), whereas those of desiccation-sensitive plants are completely destroyed after a short-term desiccation event (Deng et al. 2003).

Figure 1: Dehydration and rehydration of detached fronds of S. bryopteris.

Figure 2:Effect of dehydration and rehydration on electrolyte leakage of detached fronds of Selaginella bryopteris. The results are mean ± S.D. of three independent measurements.

About 250 protein spots were reproducibly detected and analyzed (Fig 4). It seems that proteins involved in protein destination and degradation were more expressed in desiccated fronds (Table 1). One such protein was putative Fbox/ LRR-repeat protein. This protein is a conserved domain that is present in large number of proteins with a bipartite structure. Through the F-box, these proteins are linked to the Skp 1 protein and the core of SCFs (Skp 1-cullin-Fbox protein ligase) complexes. SCFs complexes constitute a new class of E3 ligases. They function in combination with the E2 enzyme Cdc34 to ubiquitinate G 1 cyclins, Cdk inhibitors and many other proteins, to mark them for degradation. The physiological roles of proteolytic enzymes are diverse, as they are necessary both for processing proteins from an inactive to active states and for recycling redundant/ damaged polypeptides (Schwechheimer and Schwager 2004). It has been known that protein degradation via the ubiquitin–proteasome pathway plays a pivotal role in controlling cellular processes, such as cell cycle progression and transcriptional control in eukaryotic cells (Hershko and Ciechanover 1998). It is possible that induction of proteolytic enzymes, together with the upregulation of translation-related factors, is related to the biosynthesis of novel proteins involved in the drought resistance mechanisms. Rivero et al (2007) have, on the other hand, shown that suppression of drought induced senescence provided outstanding drought tolerance in transgenic tobacco plants. Two ribosomal proteins, 40S RPS27 and 60S RPL27, were up regulated under desiccation stress (Table 1). Vincent et al (2007) have reported that in grapevine shoots, there is an increased abundance of RPL39 in response to drought. In yeast, this protein is a 60S ribosomal subunit implicated in translational accuracy (Dresios et al., 2000).

Figure 3: Comparison of photosynthesis and fluorescence in the fronds of S. bryopteris exposed to dehydration and rehydration.

Figure 4: Comparison of 2D gel maps of proteins isolated from detached fronds of S. bryopteris during dehydration and rehydration. The pH range is indicated along the top of each gel, and the sizes of MW markers (kDa) are indicated down the left-hand side. U1 to U8 – up-regulated; D1 – down-regulated.

Table 1: Patterns of protein expression changes in dehydrated and rehydrated S. bryopteris fronds in comparison with that of untreated fronds; C-control, De-dehydrated, Re-rehydrated.
In the present study, a DEAD-box ATP-dependent RNA helicase 5 was highly up-regulated in dehydrated fronds and its abundance remained higher in rehydrated fronds (Table 1). DEAD-box RNA helicases have been implicated to have a function during stress adaptation processes, but their functional roles in plant stress responses remain to be clearly elucidated (Owttrim, 2006). Kim et al (2008) found differential expression in transcript levels of two RNA helicases viz. AtRH9 and AtRH25 in Arabidopsis thaliana exposed to cold, drought or salt stress. A pea DEAD-box related helicase (PDH45) transcript was induced in pea seedlings in response to a range of abiotic stresses including salt (specifically Na+), dehydration, wounding and low temperature, leading to the suggestion that pdh45 transcript accumulates in response to general water stress caused by desiccation (Sanan-Mishra et al., 2005). These results together with our result imply that these DEAD-box RNA helicases perform a crucial function and are directly involved in cellular responses to a specific abiotic stress.

The only down-regulated protein identified was photosystem I reaction center subunit III (Table 1, Fig 5). This protein participates in electron transfer from plastocyanin to P700. This protein remained under-expressed even after rehydration, although photosynthesis was restored. Sigfridsson & Oquist (2006) showed that desiccation of tolerant species such as Cladonia impexa Harm and Trebouxia pyriformis Archibald causes a preferential energy distribution into photosystem I. These plants employ this strategy to avoid photo-dynamic destruction of the photosynthetic apparatus when photosynthesis is inhibited under dry conditions. The physical properties of the photosynthetic apparatus are of crucial importance in desiccation- tolerant plants. The photosynthetic apparatus is very sensitive and liable to injury, and needs to be maintained or quickly repaired upon rehydration (Godde, 1999). At peak stress intensity, the repression of photosynthesis related genes (Rubisco small subunit, PSI reaction center subunit VI and X) may be due to stress severity and could indicate the beginning of senescence (Bogeat Triboulat et al, 2007).

Figure 5: Magnified view of some of the differentially expressed proteins. (U-upregulated; D-down regulated)

In summary, this paper has presented a primary study of the protein expression pro?le in response to dehydration and rehydration in the resurrection plant Selaginella bryopteris. Analysis of the identi?ed proteins revealed that proteins involved in protein destination and degradation were more expressed in desiccated fronds. These findings tentatively indicate that some of the proteins could contribute a physiological advantage to Selaginella bryopteris under desiccation. A more thorough study is needed with pot grown Selaginella bryopteris plants to test this speculation.

Acknowledgements
We thank Director, NBRI for his help and encouragement. This work was carried out under Supra Institutional Project (SIP-09) funded by Council of Scientific and Industrial Research, India.

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