|Farah Deeba1, Vivek Pandey1*, 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 : firstname.lastname@example.org
|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 - Response to Dehydration and Rehydration. J Proteomics Bioinform 2:108-116. doi: 10.4172/jpb.1000067|
|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.|
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Selaginella bryopteris (L.) Bak is a resurrection plant. Uniquely, its detached fronds have the ability to survive desiccation similar to that of the whole plant. In order to understand the mechanisms of desiccation tolerance, proteome studies were carried out in this plant using detached fronds 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 a drop in Fv/Fm values. Both fluorescence and photosynthesis regained totally after rehydration. About 250 protein spots were reproducibly detected and analyzed. From the putatively identified spots (proteins), it was observed that proteins involved in transport, targeting and degradation were expressed more in the desiccated fronds. These findings tentatively indicate that some of the proteins could contribute a physiological advantage to S. bryopteris under desiccation.
|Selaginella bryopteris; Desiccation tolerance; Fluorescence; Two-dimensional electrophoresis|
|Plants have evolved a wide spectrum of adaptations to cope with the challenges of environmental stress. Of the various stresses, one major factor that limits the productive potential of higher plants is the availability of water. The International Water Management Institute (IWMI), a CGIAR Institute with headquarters in Sri Lanka, predicts that by year 2025, one-third of the world’s population will live in regions that will experience severe water scarcity (http://www.iwmi.cgiar.org/). This will surely impact on agriculture in these regions. 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 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. Tolerance or resistance to this severe water deficit determines productivity of the plants, especially as food sources.|
|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, with an unique feature of detached fronds possessing a similar level of desiccation tolerance as that of whole plants. This was ascertained by studying various physiological parameters in intact plants as well as in 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 intend to use it as a model system to gain a system-level understanding of responses to desiccation using multiple platforms that provide information about global transcript levels, proteomes, a wide range of metabolites, enzyme activities, and growth parameters.
|Stress responses in plants cause changes in the structure and activity of one or more proteins. Therefore, characterizing these proteins and understanding their function is an important method for studying responses of the plants to given stress factors. Identifying and characterizing individual proteins is an enormous task that is greatly facilitated in the modern times by combining 2D-gel electrophoresis with mass spectrometry. Such a combination is a powerful tool for identifying large numbers of proteins. These techniques, in combination with the constantly expanding genomic and EST databases, also enable the simultaneous identification of a these proteins. These methods can therefore, be collectively called as the Proteome analysis methods.|
|Here, we report a proteome analysis of the changes in proteins that occur in the detached fronds of S. bryopteris, first when they are dehydrated and subsequently after they are rehydrated. Our study describes the putative identification of 9 dehydration-responsive proteins by mass spectrometry and further shows that proteins involved in transport, targeting and degradation were expressed more in the desiccated fronds than that in the original excised fronds or the re-watered fronds.|
|Materials and Methods|
|Plants of S. bryopteris were collected from wild (Mirzapur district, U.P., India; latitude 23°52´-25°32´ N & longitude 82°7´-83°33´ E) and maintained in the fern house of the Institute.
Fronds with three different water status were used in the study: Control fronds (C - these were freshly excised fronds with RWC 100%), dehydrated (S1-RWC 10%) and rehydrated(S2 - RWC 100-104%). In order to dehydrate the fronds, freshly detached fronds from well-hydrated plants were placed in petri plates and subjected to dehydration in dark at 25 °C (60% relative humidity) in a growth chamber. Control samples of detached fronds were kept fully hydrated under the same condition. Rehydration was achieved by keeping the dehydrated fronds on wet filter paper for 12 h
|Photosynthesis Parameters and Chlorophyll Fluorescence|
|Photosynthesis parameters and chlorophyll fluorescence were measured and recorded using LiCOR 6400 and PAM 2000 (WALZ) systems, respectively according to the manufacturer’s procedures. Five independent replicates were used for these physiological parameters.|
|Fronds were frozen using liquid N2, ground in frozen state in a chilled pestle and mortar to a fine powder. This powder was extracted with 50 mM Tris-HCl, pH 8.0, 25 mM EDTA, 500 mM thiourea and 0.5% 2-mercaptoethanol (BME). The extract was mixed with 10% cold TCA and 0.07% BME, and left overnight at -20 °C. The mixture was centrifuged at 4500 rpm for 10 min and the pellet was washed three times with acetone containing 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 Tris-buffered 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 °C. 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 stored at -80 °C until further use. Total protein content was analyzed using the protein assay dye reagent (Bio-Rad).|
|Isoelectric focussing (IEF) and Polyacrylamide gel electrophoresis (PAGE) Fifty μg protein was used for Isoelectric focussing (IEF) with 7 cm IPG strips, pH 4 to 7 in Ettan IPGphor unit (Amersham-GE Healthcare). 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, 0.001% bromophenol blue up to a volume of 135μl. 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 using 7 x 8 cm homogeneous 12% SDSPAGE 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 protein expression patterns were determined as up-regulated (% volume increased by two-fold or more), down-regulated (% volume decreased by two-fold or more) and unchanged (% volume varied less than or upto two-fold). Three independent replicates were used for the proteomic analyses. Only those spots present in at least three gels of independent sets were included in the analysis.|
|Tryptic digestion of the protein spots excised from the gels, and sample preparation were performed according to Koistinen et al., (2002). Briefly, gel pieces corresponding to selected spots 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 and samples were digested overnight at 37°C. Peptides were extracted twice with 50% acetonitrile containing 5% Tri-fluoroacetic acid. Gel particles were rehydrated with water, and two more extractions were performed with 50% acetonitrile containing 5% Trifluoroacetic acid. The recovered peptides were
concentrated to a final volume of 20 μl.
|The tryptic peptides were analyzed using Thermo Finnigan LCQ Advantage max ion trap mass spectrometer having Finnigan Surveyor HPLC system connected to it. The 2 μl sample was introduced into the ESI source through Finnigan Surveyor autosampler. The column was Thermo Bio-Basic 100 X 1, 5 μM and the peptides were eluted with linear 25 min gradients of 5–95% (v/v) acetonitrile with 0.1% (v/v) formic acid in water at a flow rate of 40 μl min-1. The mass spectra were scanned in the peptide mass range of 300- 1800 Da and the maximum ion injection time was set at 50 nanoseconds. Ion spray voltage was set at 5.3 KV and capillary voltage 30.5 V. The MS scan was continued up to 20 min for recording final data. The MS/MS data were processed using BIOWORKS 3.1 SR1 and searched against NCBI databases (nr Protein sequences) with the MS/MS ion searching program MASCOT (http://www.matrixscience.com) and homologies detected were scored as Ion Score (Score = –10 * log P, where P is the probability that the observed match is a random event). For the identification of proteins, Mascot search parameters were set as follows: taxonomy, Arabidopsis thaliana; fixed modification, carbamidomethylation at cysteine; variable modification, oxidation at methionine, precursor mass tolerance 1.5 Da, fragmented mass tolerance, 0.5 Da, digestion enzyme, trypsin; allowed miss cleavage, 2. Default settings were used for other parameters.|
|Results and Discussion|
Detached fronds from fully hydrated S. bryopteris were subjected to 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 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. Since the detached fronds of Selaginella also showed similar response to that of the whole desiccation-tolerant plant, C. wilmsii, our results suggest that detached fronds of S. bryopteris plant thus represents a simplified system to investigate the basis of desiccation tolerance especially by taking advantage of avoidance of possible developmental regulation and long-distance signaling from other organs.
|The fronds immediately after detachment and in a stillhydrated state showed Fv/Fm ratios around 0.8 indicating the functional photosystems (Fig 3). After dehydration the fronds showed a decrease in both net respiration and Fv/Fm ratios. Both fluorescence and photosynthesis regained totally after rehydration. This clearly showed that detached, desiccated S. bryopteris fronds fully revived their metabolism after re-hydration. In general, water deficit causes a reduction in the photosynthesis rate, resulting in a decline in the photochemical efficiency of PSII and electron transport rate in both, 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 membrane 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 even after a short-term desiccation event (Deng et al., 2003).
|In summary, this paper has presented a preliminary study of the protein expression profile in response to dehydration and subsequent rehydration using the excised frond of the resurrection plant Selaginella bryopteris. The study showed the possible involvement of proteins involved in transport, targeting and degradation were expressed more in desiccated fronds and that all expression changes were not reversed when the desiccated fronds were rehydrated. Such changes in the fronds could contribute towards a physiological advantage for withstanding potential damage that can be caused by desiccation. However, identification of more protein spots as well as in planta studies using potted whole plants of Selaginella bryopteris may be required for a better understanding of not only desiccation stress but also possible restoration of function after the plants are destressed.|
|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.|