SDS-PAGE and Two-dimensional Electrophoresis Determination of Protein from Peel and Pulp Tissues of Ripening Durian Fruit

Transcription

1 SDS-PAGE and Two-dimensional Electrophoresis Determination of Protein from Peel and Pulp Tissues of Ripening Durian Fruit A. Suwanagul and R. Deewathanawong Postharvest Technology Department Thailand Institute of Scientific and Technological Research Pathumthani Thailand Keywords: differential expression, protein profile, rind, aril, ethylene, respiration Abstract Durian (Durio zibethinus Murr.) is a tropical fruit with thick wooden skin and sharp spines covering a creamy yellow edible aril part. Ripening of the fruit is characterized by a climacteric raise of respiration and ethylene production. These physiological changes are accompanied by aril softening and various changes in chemical compositions which lead to an overall eating quality. SDS-PAGE was used to separate soluble proteins extracted from peel and pulp tissue at different ripening stages. SDS-PAGE analysis revealed a relatively similar but different in relative abundance of some polypeptide species between peel and pulp tissues during ripening. Two-dimensional electrophoreses were performed on soluble proteins from ripe and unripe peel and pulp tissues. 2D-PAGE protein profile of ripening durian peel and pulp can be characterized into six groups of polypeptide species depending on their constitutively express, significantly increase or decrease, slightly increase or decrease in their relative abundance or disappearing as the fruit become ripe. Further analysis of these ripening specific polypeptide species by either N-terminal sequencing or mass spectrometry may help to reveal a rather complex ripening behavior of this fruit. INTRODUCTION Durian (Durio zibethinus Murray) is one of the most economically important fruit crops in Thailand. Fruits varying in size, generally weigh between two to four kilograms, are characteristically covered with wooden skin with sharp spines. A creamy yellow or a yellow edible aril part of the fruit is packed inside three to five locules jointed with natural abscission layer of the skin. Each segment is enclosed with up to three reddish brown seeds. As the fruit ripened, each locule can be easily torn apart from the basal end and the aril softens. A sweet and strong cheesy, sulfurous characteristic aroma develops at this stage. Ripening of durian fruit is characterized by a climacteric raise of respiration and ethylene production (Tongdee et al., 1988; Ketsa and Daengkanit, 1998). These physiological changes were accompanied by aril softening and various changes in chemical compositions which leading to an overall eating quality of the fruit (Sriyook et al., 1994; Ketsa and Daengkanit, 1998). An undesired characteristic of fruit dehiscence was commonly found as fruit over-ripens. This has been reported to be closely implicated with fruit weight loss and various plant growth regulators, especially ethylene (Tongdee et al., 1990a; Sriyook et al., 1994). Various factors have been reported to effect respiration and ethylene production of the fruit including fruit variety, maturity stage at harvest, atmospheric gases composition (Tongdee et al., 1988; Tongdee et al., 1990b) and various postharvest treatments (Sriyook et al., 1994). Very large differences in physiology and biochemistry between pulp and peel tissue have been observed during ripening of durian fruit. Booncherm and Siriphanich (1991) reported 5 and 100 fold increase in respiration and ethylene production rate by the peel tissue than that of the pulp tissue, respectively. In more details, Tongdee et al. (1992) observed a very distinct ethylene production rate, EFE activity, ACC content between peel and pulp tissue of durian fruit during ripening at chilling temperature. More recently, Ketsa and Daengkanit (1999) and Proc. IW on TSF Eds.: N. Chomchalow et al. Acta Hort. 787, ISHS

2 Imsabai et al. (2002) have reported a changing of some cell wall degrading enzymes during durian fruit ripening. The objectives of this study was to provide an overview on the differences in soluble protein profiles from peel and pulp tissue of fruits at specific ripening stages based on their internal ethylene content using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). MATERIALS AND METHODS Mon Thong durian fruits were obtained from commercial orchards in Chanthaburi. Fruits were harvested at 110 d after anthesis and transported to the lab within the same day. Respiration and ethylene production measurement were done by placing an individual fruit in a sealed container for 1 h at 22 o C. One ml gas sampling was taken from each container through a rubber sampling port for determination of CO 2 evolution and ethylene production. Internal gas sample was taken from each fruit using a method modified from Saltveit (1982). A piece of rind was carefully removed from each fruit with sterile 0.6 mm cork borer without damage the aril. The hole was fitted with a short plastic tube and seal to the rind with synthetic cement. The other side of the tube was tightly plugged with a septum. A ml gas sample was then taken from the septum for CO 2 and C 2 H 4 analysis. CO 2 and C 2 H 4 concentration were analyzed by gas chromatograph (Shumadzu Model 9A) equipped with TCD and FID, respectively. A total of six ripening stages plus two sub-stages were defined according to an internal ethylene level described in Fig. 1. Ripening stage 1 refer to un-ripe fruit where the lowest internal ethylene concentration (below 0.1 ppm) is detected. Fruits at this stage of ripening are inedible. Stages 2 and 3 refer to fruit at on set of climacteric where the internal ethylene concentration began to increase from ppm, respectively. Fruits at these stages are stilled inedible. Ripening stage 4 refer to ripe fruit where the highest internal ethylene level (above 3.5 ppm) is detected. Fruits at this stage are in a perfect stage for consumption. Ripening stage 6 refer to fruit at over-ripe stage where internal ethylene concentration receded (below 2.3 ppm). Fruits at this stage are not suitable for fresh consumption and can be used only for processing. Pulp and peel tissue of durian were collected from the fruit at each specific ripening stage. An exact amount of each tissue was sliced, pre-weighted and wrapped in aluminum foil, dipped in liquid nitrogen and stored at 80 o C until used. Soluble protein was extracted from pulp and peel tissue of durian using the method described by Coleman et al. (1991) with a minimal modification. Frozen pulp and peel tissues (10 g fresh weight each) were ground into a fine powder in liquid nitrogen using a coffee grinder. The ground tissue was then homogenized at 4 o C with a homogenizer (CAT Model X-520) for 1 min at maximum speed in 8 volumes (w/v) of extraction buffer ( 50 mm sodium borate, 50 mm ascorbic acid, 1% β-mercaptoethanol (ph 9.0); 1 mm PMSF was added before homogenizing). The sample was then centrifuged for 30 min at 4 o C and 35,000 g. Five volumes of 0.1 M ammonium acetate in 20 o C methanol was added to the supernatant and held at 20 o C overnight to precipitate proteins. The precipitate was collected by centrifugation at 10,000 g for 20 min at 20 o C. The protein pellet was then washed three times with 0.1 M ammonium acetate in 20 o C methanol and once with 20 o C acetone before air dried. The protein pellet was resuspended in Laemmli (1970) s lysis buffer or in 4% CHAPS buffer (if to be used for IEF), held in boiling water for 5 min and cooled to room temperature before storage at 20 o C until used. Protein content was determined according to the method described by Bradford (1976), using Bio-Rad dye binding protein assay (Bio-Rad). Bovine serum albumin (BSA) ranging from 20 to 100 µg ml -1 was used as a standard protein. SDS-PAGE was performed according to the method described by Laemmli (1970). Total soluble protein (50 µg) were loaded on 12.5% SDS-PAGE slab gel (18 x 16 cm) with 4% stacking gel and electrophoresed at 25 ma per gel for 5 h. Gels were stained with 0.1% Coomassie blue R-250 in 40% methanol 5% acetic acid and destained in the 216

3 same solution lacking Coomassie blue. The MW-SDS-70 (Sigma) was used as a molecular weight marker. Gel pictures were taken with Kodak DC120 digital camera and were analyzed with Kodak Digital Science 1D Image Analysis Software (Kodak). For the isoelectric focusing (IEF), 15 µg of total soluble protein was loaded on to 3-10 non-linear ph gradient IPG strip (13 cm in length) and separated with IPGphor Isoelectric Focusing System (Amersham Biosciences) according to a manufacturer s manual. For the second dimensional electrophoresis, the focused strip was placed on 12.5% SDS-PAGE slab gel (18 x 16 cm) without stacking gel and electrophoresed at 15 ma/gel for 15 min followed by 25 ma/gel for 5 h. Gels were stained with PlusOne protein silver staining kit (Amersham Biosciences). Gel pictures were taken with Kodak DC120 digital camera and were analyzed with Image Master 2D Software (Amersham Biosciences). Isoelectric point and molecular weight of the separated polypeptides were estimated based on the relative length of IPG strips to their ph gradient and a relative mobility of MW-SDS-70 molecular weight marker. RESULTS AND DISCUSSION SDS-PAGE analysis of total protein extracted from peel and pulp tissue at different ripening stages are showed in Figure 2 and 3, respectively. A comparison between the two profiles revealed a relatively similarity but different in relative abundance of polypeptide species from peel and pulp tissue of durian during ripening. Almost all polypeptides within the two profiles appeared to be constitutive expressed in all ripening stages. The protein profile of ripening pulp tissue is characterized by the most abundance polypeptide with a molecular weight of 47.5 kd (Fig. 2). This polypeptide species started to increase in relative abundance at early stage of fruit ripening (stage 2) and continue expressed at high level until the end of ripening period. Since there was a limit amount of this polypeptide found in peel tissue, therefore, one can expected this polypeptide may played a major role in durian pulp softening. The protein profile of ripening peel tissue can be characterized by the two most abundant polypeptide species with a molecular weight of 28 and 53 kd (Fig. 3). While the expression of a 53 kd polypeptide appeared to be steady or slightly increased as fruit ripening, a significant increase in relative abundance of the 28 kd polypeptide was observed as fruit began to ripen (ripening stage 2 to stage 3.5). A remarkable increase in the abundance of the 28 kd polypeptide was found in fruit at ripening stage 4. Fruits at this stage of ripening became eating-ripe and exhibited the highest internal ethylene level. Interestingly, the expression of this polypeptide still remained at high level as ripening progressed through stage 5 or 6 where internal ethylene content has started to recede. Therefore, it is doubtful that this 28 kd polypeptide may or may not be directly involved in ethylene biosynthesis of the fruit. Two-dimensional protein pattern of pulp at un-ripe stage (A, ripening stage 1) and eating-ripe stage (B, ripening stage 4) is shown in Figure 4. A protein profile of ripening durian pulp tissue can be characterized by approximately five groups of polypeptide species. The first two groups of polypeptides appeared to be constitutive and/or having a slight increase in their relative abundance as the fruit became ripe. This group of polypeptides include polypeptide species within group a and group c, with an estimated molecular weight of 74 and 39 kd, respectively. The third group of polypeptides was found to have a significant accumulation as the fruit became to ripen. This group of polypeptide is included three to four polypeptide species within group b, which having an estimated molecular weight of 47.5 kd. The forth group of polypeptide was found to decrease in their relative abundance as the fruit became to ripen. This group of polypeptide is included polypeptide species within group d and group e, with an estimated pi of 5.5 and 5.5 to 6.2, respectively. The last group of polypeptides was observed to disappear as the fruit ripens. This group of polypeptides includes polypeptide species within group f, group g and group h. Fig. 5 showed two-dimensional protein pattern of durian peel tissue at un-ripe 217

4 stage (A, ripening stage 1) and eating-ripe stage (B, ripening stage 4). The protein pattern of ripening peel tissue can be characterized by four groups of polypeptide species as indicated by small letters and oval marks on the gel. The first group of polypeptides (group c) with a molecular weight of 43 kd appeared to be constitutive expressed in both un-ripe and ripe peel tissue and can be used as a marking point to facilitate a comparison between those two profiles. The second group of polypeptides contained two groups of polypeptide species (group a and group b) with a molecular weight of about 53 kd. These two groups of polypeptide species appeared to have a slight decrease in relative abundance as the fruit ripen. The last two groups of polypeptide species (group d and group e) may be the most characteristic of two-dimensional electrophoresis of proteins from peel tissue. Whereas, a significant increase in relative abundance was found in a group of polypeptides species (group e) with a molecular weight of 28 kd, a significant decrease in relative abundance was found in a group of polypeptides with a molecular weight between 36 to 39 kd and isoelectric point (pi) of 3.5. Thus, these two groups of ripening specific polypeptide species are likely to play a key role in durian fruit ripening and may be of most importance to undertaking further investigations. A very distinctive protein pattern has been reported between peel and pulp tissues of apple during fruit maturation (Lara and Vendrell, 2000) and more recently, during cold storage (Lara and Vendrell, 2002). Those differences have been implicated in a complex regulation of ethylene biosynthesis enzyme, specifically 1-aminocyclopropane-1- carboxylate synthase (ACC synthase) and 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase), in peel and pulp tissues of the fruit. From the most recent results from our cloning and characterization of ripening related genes using mrna from this set of fruit sample, two full-length clone of genes in ethylene biosynthesis, ACC synthase and ACC oxidase were successfully cloned, GenBank accession EF and EF101897, respectively. The deduced amino acid sequence of ACC synthase and ACC oxidase clones suggested an encoded polypeptide with the molecular mass of 55.4 kd and 36 kd, respectively. By comparing a deduced molecular mass with protein profiles from SDS- PAGE and 2D-PAGE, it has the possibility that a polypeptide(s) which having an apparent molecular weight of 53 kd (Fig. 3 and 5a) and a group of polypeptides which having an apparent molecular weight between kd (Fig. 5d) might related to ACC synthase and ACC oxidase, respectively. However, it is still far too early to relate this observation with any biological regulatory processes. More research, either by N-terminal sequencing or determination of molecular mass by mass spectrometer after 2D-PAGE is required for identification and characterization of these differentially polypeptides from peel and pulp tissues. Literature Cited Boonchern, P. and Siriphanich, J Postharvest physiology of durian pulp and husk. Kasetsart J. Nat. Sci. 25: Bradford, M.M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle-dye binding. Ann. Biochem. 72: Coleman, G.D., Chen, T.H.H., Ernst, S.G. and Fuchigami, L Photoperiod control of poplar bark storage protein accumulation. Plant Physiol. 96: Imsabai, W., Ketsa, S. and van Doorm, W.G Effect of temperature on softening and the activities of polygalacturonase and pectinesterase in durian fruit. Post. Biol. Technol. 26: Ketsa, S. and Daengkanit, T Physiological changes during postharvest ripening of durian fruit (Durio zibethinus Murray). J. Hort. Sci. Biotech. 73: Ketsa, S. and Daengkanit, T Firmness and activities of polygalacturonase, pectinesterase, B-galactosidase and cellulose in ripening durian harvested at different stages of maturity. Sci. Hort. 80: Lara, D. and Vandrell, M Changes in abscisic acid levels, ethylene biosynthesis, and protein patterns during fruit maturation of Granny Smith apples. J. Amer. Soc. Hort. Sci.125:

5 Lara, D. and Vandrell, M Cold-induced ethylene biosynthesis is differentially regulated in peel and pulp tissues of Granny Smith apple fruit. Post. Biol. Technol. 29: Laemmli, U.K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: Saltveit Jr., M.E Procedures for extracting and anlysing internal gas samples from plant tissue by gas chromatography. Hort. Sci. 17: Sriyook, S., Siriatiawat, S. and Siripanich, J Durian fruit dehiscence-water status and ethylene. Hort. Sci. 29: Tongdee, S.C., Suwanagul, A. and Neamprem, S Harvesting maturity in relation to ripening in tropical fruit. p In: S. Maneepun and P. Varangoon (Eds.), Food Science and Technology in Industrial Development. Vol. 2. Proc. Food Conference 88. Bangkok, Thailand. Tongdee, S.C., Suwanagul, A., Neamprem, S. and Bunreungsri, U. 1990a. Effect of surface coatings on weight loss and internal atmosphere of durian. ASEAN Food J. 5: Tongdee, S.C, Suwanagul, A. and Neamprem, S. 1990b. Durian fruit ripening and effect of variety, maturity stage at harvest and atmospheric gases. Acta Hort. 269: Tongdee, S.C., Bonruengsri, U. and Neamprem, S Effect of storage temperature on ethylene biosynthesis and carbon dioxide production in the peel and the pulp of ripening durian fruit. Proc. the 30 th Kasetsart Uni. Ann. Conference, Bangkok, Thailand. 29 Jan.-1 Feb. 1992, p

6 Figures Fig. 1. Internal ethylene level and ethylene production rate (A) and internal carbon dioxide level and respiration rate (B) of durian cv. 'Monthong' at different ripening stages. Each parameter within the same ripening stage was obtained from the same representative fruit. Symbol within the bracket indicated a subjective assessment of pulp condition: (-) = firm, inedible; (-+) = partially firm (yielded to fingers), inedible; (+) = partially soft, optimum eating quality; (++) = soft, optimum eating quality; and (+++) = very soft, over-ripen. 220

7 47.5 Fig. 2. SDS-PAGE profile of total protein extracted from pulp tissue of Mon Thong durian at different ripen- ing stages. Ripening stages are defined as numbers (1-6) according to materials and methods. The molecular weight of protein standards is indicated on the left. Lanes marked with letter M are protein standards. Arrow indicate a position of protein species which is more or less abundant between ripe (R) and unripe (UR) fruit tissue. 221

8 53 28 Fig. 3. SDS-PAGE profile of total protein extracted from peel tissue of Mon Thong durian at different ripening stages. Ripening stages are defined as numbers (1-6) according to materials and methods. The molecular weight of protein standards is indicated on the left. Lanes marked with letter M are protein standards. Arrow indicate a position of protein specie which is more or less abundant between ripe (R) and unripe (UR) fruit tissue. 222

9 MW (kd) a b A pi 45 d c e 31 f g h pi 97.4 a B 66.2 b 45 d c e 31 f g h Fig. 4. Two-dimensional protein pattern of Mon Thong durian pulp tissue at unripe stage (stage 1, A) and eating-ripe stage (stage 4, B). Estimated isoelectric point (pi) and molecular weight (MW) are indicated on top and left hand side of each gel, respectively. A polypeptide or a group of polypeptides which most represent each gel is enclosed in an oval and marked with a small letter. 223

10 MW (kd) A. PI d a b c 31 e MW (kd) B. PI d a b c 31 e Fig. 5. Two-dimensional protein pattern of Mon Thong durian peel tissue at unripe stage (stage 1, A) and eating-ripe stage (stage 4, B). Estimated isoelectric point (pi) and molecular weight (MW) are indicated on top and left hand side of each gel, respectively. A polypeptide or a group of polypeptides which most represent each gel is enclosed in an oval and marked with a small letter. 224