Two dimensional electrophoresis of thylakoid membrane proteins of wheat seedlings under UV-B irradiation: comparison of two wheat cultivars.

Transcription

1 Chapter 4 Two dimensional electrophoresis of thylakoid membrane proteins of wheat seedlings under UV-B irradiation: comparison of two wheat cultivars. Contents 4. I Introduction 4.2 Materials and methods Plant material, growth condition and UV-B treatment Isolation ofthylakoid membranes D electrophoresis Solubilization ofthylakoid membranes Isoelectric focusing (IEF) in first dimension SDS-PAGE in second dimension ph gradient determination Gel staining 4.3 Results 4.4 Discussion 33

2 4.1 Introduction In higher plants induction of unique proteins m response to high temperatures is a feature common to all species. These novel proteins termed HSP (heat shock proteins) (Clarke and Critchley, 1990), play an integral part in the heat response. Similarly when the thylakoid polypeptide analysis by SDS PAGE was done to look for certain proteins which gets triggered in UV-B stress, no major difference was found in the protein profiles in single dimensional PAGE except a 14 kda unknown polypeptide with contrasting behavior in two wheat cultivars (see chapter 3, Fig. 3.3 and 3.4 ofthis thesis). Two dimensional (2-D) gel electrophoresis is a powerful technique devised for the separation and characterization of proteins. This technique was originally developed by O'Farrell in 1975 (O'Farrell, 1975) and was used for protein identification and purification (Celis and Bravo, 1984). With recent progress in research on the thylakoid membrane organization, more sensitive and comprehensive methods such as 2-D and its associated satellite techniques for membrane protein separation and identification are required besides polyacrylamide gel electrophoresis (I-D). Since, the 2-D approach permits the determination of not only molecular weight, but also isoelectric points with extreme precision. By this method isoforms of the protein can also be distinguished. Relatively few reports regarding 2-D electrophoresis of thylakoid membrane proteins can be found in the literature, because of serious streaks over the second-dimensional protein pattern due to inefficient removal of chlorophylls from the proteins and incomplete solubilization. As mentioned elsewhere (Roscoe and Ellis, 1982; Remy and Ambard Bretteville, 1985) thylakoid membrane proteins are very difficult to solubilize smce photosynthetic apparatus is composed of multiple hydrophobic chlorophyll and carotenoid-binding proteins. The aim of using this technique for separating thylakoid proteins was to increase the resolution of the myriad proteins found in thylakoids above that obtained with one-dimensional system. Yu eta/., 1994 presented a modified 2-D technique which can be employed for the identification and isolation of polypeptides of the thylakoid membrane and for its direct application to microsequencing analysis. In an effort to further the information regarding the effect of UV -B stress on thylakoid proteins, in this work additional evidence for heterogeneity of the thylakoid membrane organization at the molecular level is provided. 111

3 4.2 Materials and Methods Plant material, growth condition and, UV-B treatment Wheat seeds (Triticum aestivum L. cv. HD2329 and K65 obtained from Indian Agriculture Research Institute, New Delhi) were surface sterilized with 0.1% HgCl2 solution. They were germinated on moist filter paper in dark at 25 C for 48 h. After germination, the seedlings were transferred to a plant growth chamber maintained at 25 C arid 14 h/10 h, light/dark cycles. Light intensity (cool white fluorescent lamps) during growth of the seedlings was E m-2 s-1. The seedlings used for all experiments were 8 d old. The UV -B stress treatment was started 3 h after onset of photoperiod. UV -B treatment to wheat seedlings was given in a plant growth chamber maintained at 25 C, specially fitted with a single UV-B source, supplying UV-B with maxima at 312 nm. Wheat seedlings were subjected to 5 h of supplementary UV -B (1 00 ~-te m-2 s-1 of white light and 40!J.E m-2 s-1) and UV-B (40 ~te m-2 s-1). The leaves from irradiated seedlings were used for isolation of thylakoid proteins Isolation of thylakoid membranes The wheat leaves were cut into pieces with a pair of sharp scissors. The chopped leaves were blended in semi-frozen isolation buffer containing I 0 mm N~P207, 4 mm Mgcl2, 2 mm ascorbate, 0.33 M sorbitol ph 6.5. A cocktail of protease inhibitors (antipain, PMSF, aprotinin, bestatin, EDT A, leupeptin, pepstatin) was included in homogenization buffer. The resulting slurry was filtered through eight layers of cheese cloth and the filtrate was centrifuged in a refrigerated centrifuge (Hitachi CR20B2 Tokyo, Japan) 250 x g for 1 min. to remove the cell debris. The supernatant was again centrifuged at 4000 x g for 5 min. The pellet was suspended in resuspension buffer containing 50 mm Hepes (ph 7.6), 2 mm EDT A, 1 mm MnCI2, 0.33 M sorbitol and 0.1% (w/v) BSA. All the steps were carried out in dark at 4 C. Chi concentration was determined according to the method of Arnon, ( 1949) D electrophoresis (Roscoe and Ellis, 1982) I Solubilization of thylakoid membranes To produce consistent 2-D patterns of thylakoid proteins with sharp spots and a minimum of streaking, it was necessary to first remove the lipids with 35

4 acetone. The thylakoid membrane fraction ( approx. 400 j..tg) was washed twice by suspending it in 10 ml resuspension buffer (25 mm Tricine-KOH (ph 8.0), containing 5 mm ~-mercaptoethanol) and sedimentation at 20,000 x g for 10 min at 4 C. The washed pellet was taken up in I ml resuspension buffer to produce a homogenous suspension to which 9 ml acetone was added at room temperature with mixing. It is important to add acetone to a homogenous suspension of thylakoids, and to avoid addition of acetone directly to thylakoid pellets. The protein was allowed to precipitate by standing on ice in dark for 60 min. The protein was pelleted by centrifugation at 5000 x g for 15 min at 0 C. The pellet was washed by addition of 10 ml acetone to produce a homogenous suspension. The precipitate was sedimented by centrifugation at 5000 x g for 15 min at 0 C. The pellet can then be stored at -20 C at this point. The white thylakoid pellet was suspended by addition of 25 ~-tl 5% (v/v) SDS at room temperature and solubilized as much of suspension as possible using a micropipette. The suspension was then placed in boiling water bath for two minutes. It was cooled to room temperature under running water. The concentration of SDS was reduced to I% by addition of 100 j..tl of Solution A which contain urea and nonionic detergent. The suspension was then cleared by centrifugation at 10,000 x g for 5 min in a microfuge to remove undissolved material. The supernatant fraction was removed and used at once for loading on IEF gels. Supernatant fraction can be stored at -20 C for at least I 0 days with no apparent effect on the resulting 2-D pattern Isoelectric focusing (IEF) in first dimension Acrylamide stock (10 ml) Acrylamide gm 36

5 Bis-acrylamide gm Make to 10 ml with deionized H20 Preparation of gel 2.65 ml of gel-mix was prepared by adding Urea 2.75 gm NP40 Deionized H20 Acrylamide Ampholines (BIORAD) APS TEMED 1 ml (1 0% stock) 985 ~1 665 ~1 (28.38% w/v acrylamide; 1.62% bis-acrylamide) 80 ~1 (1.6% ph range 5-7) 20 ~1 (0.4% ph range ) 5 ~1 (10% stock) 5 ~1 Solution A (5 ml) Urea (9.5 M) 2.88 gm NP40 (2%) 100 ~1 (1 0% stock) P-mercaptoethanol (5%) 250 ~1 Ampholines (1.6%) 80 ~1 (ph range 5-7) (0.4%) 20 ~1 (ph range ) Make to 5 ml with deionized H20 Solution B (5 ml) Urea (5 M) 1.5gm Ampholines (0.8%) 40 ~1 (ph range 5-7) (BIORAD) (0.2%) 10 ~1 (ph range ) Make to 5 ml with deionized H20 IEF was carried out in 18 em glass tubes with an internal diameter of 1. 5 mm. Each tube was treated with chromic acid before use and the gel was poured to a length of 11 em. The gel was overlayed with 40 ~I of 8 M urea and allowed to polymerize for at least 60 min. The overlay was removed, and replaced with 40 Jll of 37

6 Solution A. This in turn was overlayed with deionized water and left for further 60 min. The entire overlay was removed, and the gels were transferred to Protean II apparatus (BIORAD). 40 Ill of Solution A was added to the top of the gel, and overlayed with 0.02 M NaOH. The upper reservoir was filled with M NaOH and lower reservoir with 0.01 M H3P04. The gels were preelectrophoresised for 15 min at 200 V, for 30 min at 300 V, and then for 30 min at 325 V. The upper reservoir buffer and overlay was then discarded and samples were loaded on the tubes with 400 IJ.g protein per gel and overlayed with 40 IJ.l Solution B. The tubes and upper reservoir are filled with 0.02 M NaOH, and electrophoresis carried out for h at 325 V at room temperature. The gels were removed from tubes by injection of distilled water from a syringe between the gel and the glass. The gel can be stored frozen at -20 C for a week wrapped in parafilm or used at once for SDS gel electrophoresis SDS-PAGE in second dimension The second dimension involves Tricine SDS-P AGE electrophoresis. Tricine was used as the trailing ion, which allows resolution of small proteins at lower acrylamide concentrations than in Glycine SDS-PAGE system. The 10%T, 3%C gel without urea was useful for the range from 5 to 100 kda. It was overlayed with a 4%T, 3%C stacking gel (Schagger and Jagow, 1987). The gel was cast between glass plates one of which was ground away to produce a V -shaped groove in which the first dimension gel was supported (Protean II, BIORAD). Frozen IEF gels were allowed to warm at room temperature, and positioned on the SDS slab gel, and sealed into place avoiding the bubbles with a small amount of 1% agarose (prepared in upper reservoir buffer). After the agarose has set, SDS-denatured mol.wt. marker protein are loaded in a side slot as required. 38

7 ph-gradient determination An isoelectric focusing gel containing no sample was cut into 1 em pieces and placed in a vial containing 1 Qml of 0.1 N KCL overnight at 4 C. After that, the ph value of each vial was measured and standard curve was set up. Then based on the standard curve, the pi of the polypeptides in the 2-D protein Pattern was ascertained Gel staining The gels were first fixed in 50% Methanol and 10% Glacial acetic acid and then stained with Brilliant Blue G (0.025% in 10% Glacial acetic acid). The stained gels were destained in 10% Glacial acetic acid. 4.3 Results The aim of the present study was to examine changes in protein profiles due to supplementary UV-B and UV-B treatment, as a step towards identification of proteins that might potentially be involved in process of UV-B mediated degradation or an increase/decrease in their amounts. Figure 4. 1 shows the acetone precipitated thylakoid protein profiles of two wheat cdtivars HD2329 (panel HD) and K65 (panel K). Comparing these profiles with that of thylakoid protein profiles in chapter 3 Fig It is clear that the bands are more sharp due to removal of lipids and chlorophylls. The 2-D patterns of thylakoid proteins from the HO-C control, 5 h supplementary UV-B (panel HD-5 h Sup) and 5 h UV-B (panel HD-Sh UV-B) treated samples were compared using the same amount of protein (Fig. 4.2). There were many obvious differences in the relative intensities of polypeptide spots on the gels that correspond to 5 h Sup and 5 h UV-B. The most apparent change was an increase in intensities of spot 1 and 2 after supplementary UV -B and UV -B treatment. Whereas decrease in intensities at a cluster of spots (no. 3 & 4) were also apparent on supplementary UV-B and UV-B treatment. Less streaking was observed in panels HD-5 h Sup and HD-5 h UV-B. Similarly on comparing the 2-D patterns of thylakoid proteins from the K-C control, Sh supplementary UV-B (K-5 h Sup) and 5 h UV-B (K-5 h UV B) treated samples (Fig. 4.3), Contrary to the K-C control the protein abundance of spots no 1 and 2 decrease in supplementary UV -B and UV -B treatment. Comparing the K-C and 5 h Sup, 2-D pattern, there is an increase in abundance of many other protein in supplementary UV -B where as in only UV B the number of spots on the gel were reduced.

8 --HD-- --K-- kda : Fig. 4.1 Tricine SDS-P AGE (I 0% T, 3%C) of acetone precipitated thylakoid proteins. Two wheat cultivars HD2329 (Panel HD) and K65 (Panel K) proteins are shown. The sharpness of bands after precipitation show that apparently most of lipids, chlorophylls and carotenoid pigments were removed.

9 HD-ShSup. koa PH7 HD-ShUV-8 Fig. 4.2 The two-dimensional electrophoresis patterns of thylakoid proteins of wheat cultivar H Each sample (400 Jlg) was separated by 2-0 electrophoresis. Horizontal, isoelectric focusing (IEF); vertical, Tricine SOS-PAGE (Coomassie Brilliant blue-g stained 10% T, 3%C polyacrylamide gel). Panel HO-C, 8-days old control seedlings; Panel H0-5 h SUP, supplementary UV-B treated wheat seedlings; Panel H0-5 h UV-B, 5 h UV-B treated wheat seedlings. Arrows in H0-5 h Sup and H0-5 h UV-B indicate the decrease or increase in relative intensities with respect to HO-C.

10 koa & ph4... Fig. 4.3 The t\vo-dimensional electrophoresis patterns of thylakoid proteins of wheat cultivar K65. Each sample (400 ~g) was separated by 2-D electrophoresis. Horizontal, isoelectric focusing (IEF); vertical, Tricine SDS-PAGE (Coomassie Brilliant blue-g stained 10%T, 3%C polyacrylamide gel). Panel K-C, 8-days old control seedlings; Panel K-5 h SUP, supplementary UV-B treated wheat seedlings; Panel K-5 h UV- 8, 5 h UV-B treated wheat seedlings. Arrows in K-5 h Sup and K-5 h UV-B indicate the decrease or increase in rel ative intensities with respect to K -C.

11 4.4 Discussion 2-D gel electrophoresis 'is used to analyze and compare the effect of environmental changes on many proteins (Bewley ID, et al., 1983; Clarke and Critchley, 1990). The effect of phosphate deficiency in ~aize on the 2-D electrophoretic patterns of soluble proteins were evaluated in maize by Usuda and Shimogawara, To compare 2-D gels, it is essential that the proteins are well resolved, the gels are substantially free of streaking, smearing, and background staining, the gels lack artifacts due to proteolysis and that protein patterns are reproducible from gel to gel. Although the 2-D separations of protein solubilized from whole plant tissues (Bewley et al., 1983; Des Francs et al., 1985; Zurfluh and Guilfoyle, 1980; Clarke and Critchley, 1990) or from in vitro translation reactions (Theologi and Ray, 1982) meet this criteria, plant membrane proteins seem to be resistant to 2-D gel analysis (Brooz and Travis, 1980). Duet<? these problems the gels shown in thi~ chapter also have streaking and smearing, probably as a re.sult of incomplete disruption of all protein complexes and aggregates during solubilization, but nevertheless the 2-D gels shown here at least are able to show the effect of supplementary UV -B and UV -B on wheat thylakoid polypeptides. The careful study of thylakoid proteins using 2-D gel electrophoresis described in this chapter clearly demonstrates that effect of supplementary UV B and UV -B on wheat thylakoid protein is more complex than can be seen with single-dimension electrophoresis. Significant difference in number as well as the intensity of a number of spots can be seen. Supplementary UV -B and UV -B stress affect the thylakoid membrane protein to a large extent and thus the photosynthetic apparatus. 40