harvest and at severa1 softening stages following a postharvest ethylene treatment. .... at different stages of ripeness were assayed for XET activity. MATERIALS ...
Plant Physiol. (1993) 103: 1399-1406
Xyloglucan Endotransglycosylase Activity lncreases during Kiwifruit (Actinida d e k o s a ) Ripening' lmplications for Fruit Softening Robert J.Redgwell' and Stephen C. Fry* Centre for Plant Science, Division of Biological Sciences, Daniel Rutherford Building, The University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JH, United Kingdom
The loss of firmness in many fruits that occurs during ripening is thought to result from enzymic modification of the cell wall, causing a net loss of some structural components. A reassessment of the relationship between cell wall degradation and fruit softening has been fueled by the discovery that PG is not the primary determinant of softening in tomato (Smith et al., 1988; Giovannoni et al., 1989). Furthermore, when the cell wall pectic polysaccharides of softening fruit are isolated using procedures that minimize their inadvertent breakdown, it is found that depolymerization of the pectic backbone in vivo is less than previously
reported (Huber, 1992; Redgwell et al., 1992). The de-emphasizing of the role of PG and the depolymerization of pectic polysaccharides in fruit softening has focused attention on the nonpectic components of the fruit cell wall, cellulose and hemicelluloses, and the enzymes that act on them. In kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var deliciosa cv Hayward), cell wall changes during softening are characterized by solubilization of the pectic polysaccharides, loss of Gal from the side chains of the pectin molecules, a decrease in the M,of xyloglucan, and a dramatic swelling of the cell wall (MacRae and Redgwell, 1992). It has been suggested that wall swelling may be caused by changes in the cellulose-hemicellulose interaction and that swelling or loosening of the wall may itself be a factor in the release or solubilization of pectic polysaccharides (Redgwell et al., 1991). Avocado and strawberry, two fruits where cell wall swelling is pronounced during ripening, show a major increase in cellulase activity as the fruit soften (Christoffersen et al., 1989; Abeles and Takda, 1990). Although in theory cellulase can act on the D-(ld)-backbone of both cellulose and xyloglucan, studies with avocado (O'Donoghue and Huber, 1992) have demonstrated that C,-cellulase was not involved in ripening-related depolymerization of xyloglucan. To date there is a lack of published information on cell wallassociated enzymes in ripening fruit that can specifically modify xyloglucan. However, the recent discovery of the enzyme XET (McDougall and Fry, 1990; Smith and Fry, 1991; Fry et al., 1992b; Nishitani and Tominaga, 1992)has provided a nove1 candidate for consideration as a factor in cell wall changes leading to fruit softening. XET catalyzes both the endo-type splittirrg of a xyloglucan molecule and the linking of the newly generated (potentially) reducing end to a nonreducing end of another xyloglucan molecule. The favored role of XET is as a reconnecting enzyme involved in the rearrangement of xyloglucans to accommodate chemical creepage during cell growth. Support
Supported in part by a grant from the New Zealand Kiwifruit Marketing Board and in part by a European Community "BRIDGE" contract. Present address:Horticultureand Food Research Institute of New Zealand Ltd, Private Bag 92169, Auckland, New Zealand. * Corresponding author; fax 44-31-650-5392.
Abbreviations: CWM, cell wall material; IP, inner pericarp; kgf, kilogram-force required on penetrometer; OP, outer pericarp; PAW, pheno1:aceticacid:water (2:1:1, w/v/v); PG, polygalacturonase (EC 3.2.1.15); SSC, soluble solids concentration; XET, xyloglucan endotransglycosylase;XXXG, xyloglucan-derivedheptasaccharide (see Fry et al., 1993).
l h e activity of xyloglucan endotransglycosylase (XET) was assayed in three tissue zones of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var deliciosa cv Hayward) at harvest and at severa1 softening stages following a postharvest ethylene treatment. At harvest, extractable XET activity per unit fresh weight i n the inner pericarp (IP) and core tissue was 4.5 and 42 times higher, respectively, than in the outer pericarp (OP). Within 24 h of ethylene treatment there was an increase in the adivity and specific activity of XET in all tissues that continued throughout softening. Activity increased most in the OP, where it showed a 12-fold rise 6 d after ethylene treatment compared with 4.5- and 2.5-fold increases in the IP and core tissues, respectively. Visible swelling of the cell wall in each tissue was observed 24 h after the first detectable rise i n XET activity and was most pronounced in the OP, which showed the greatest percentage increase in XET activity. Xyloglucan, galactoglucomannan, and cell wall materials isolated and purified from kiwifruit OP were tested as donor substrates for kiwifruit XET. The enzyme showed activity against xyloglucan but was inadive against galadoglucomannan. XET was active against cell wall materials from unripe and ripe fruit, with swollen walls from the latter being the better substrate. l h e results indicate that XET may have a key role early in fruit ripening, loosening the cell wall i n preparation for further modification by other cell wall-associated enzymes.
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for this idea is provided by a strong correlation between growth rates and XET activity in spinach cell cultures and pea stems (Fry et al., 1992a), especially after GA3 treatment (Potter and Fry, 1993). Nevertheless, in the presence of suitable oligosaccharides (such as could arise during partia1 lysis of matrix polysaccharides during fruit ripening), XET can catalyze polysaccharide-to-oIigosaccharideendotransglycosylation. Unlike the polysaccharide-to-polysaccharide endotransglycosylation postulated to occur in rapidly growing cells, polysaccharide-to-oligosaccharide endotransglycosylation would cause a reduction in the M, of the xyloglucan (Farkai et al., 1992; Lorences and Fry, 1993) and a permanent loosening of the wall. Evidence that tomato contains a specific xyloglucanase that is ‘activated” by xyloglucanderived oligosaccharides has been reported (Maclachlan and Brady, 1992). It is probable that this enzyme is an endotransglycosylase. In the present study the OP, IP, and core tissues of kiwifruit at different stages of ripeness were assayed for XET activity. MATERIALS AND METHODS Plant Material
Kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var deliciosa cv Hayward) were obtained from the Ente Regionale di Sviluppo Agricolo experimental farm at Beano di Codroipo, Udine, Italy. They were picked at a very unripe stage (mean SSC 5.8%) and sent to Edinburgh under normal flight conditions. Six days after picking, when the mean SSC was 6.9%, the fruit were treated with ethylene (1000 ppm, 12 h, 25OC) and stored at 25OC after ethylene treatment. Samples of fruit were taken before ethylene treatment (harvest) and at 1, 2, 4, 5, and 6 d after treatment, i.e. at progressive stages of softness. A sample was also taken from fruit stored for 15 d at 25OC without an ethylene trea tment . For each ripening stage a single 10-fruit sample was taken. Each fruit was assessed for percent SSC (g soluble solids/100 g juice) with an Atago refractometer. The refractometer was calibrated with water (0% SSC) and a solution containing 6 g of Suc in 94 g of water (6% SSC). Flesh firmness (kgf) was measured with an Effigi penetrometer after remova1 of a 1mm-thick dlsc of skin from each side of the fruit (Lallu et al., 1989). Fruit were peeled and the OP, IP, and core tissue zones were cut into 2 cm3 pieces, frozen in liquid nitrogen, and stored at -8OOC pending extraction. Pea (Pisum satiuum L. cv Alaska) stems (20 g) were collected from etiolated plants 12 d after germination, cryo-milled in liquid nitrogen, and stored at -8OOC.
Plant Physiol. Vol.
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Extraction and Assay of XET
Approximately 10 g of frozen tissue pieces were C ~ O milled to a powder in liquid nitrogen. Samples (2 g) plus 150 mg polyvinylpolypyrrolidone were homogenized with a Polytron in 6.0 mL of 0.25 M Mes (Na+)buffer (pH as stated for individual experiments), 0.4 M NaC1, 10 mM sodium tetrathionate. The last was added to inhibit the protease, actinidin (Boland and Hardman, 1972). The homogenate was centrifuged (2000g, 10 min) and the supematant was assayed for XET. Although the extracts were not desalted, the data in Table I1 suggest that kiwifruit extracts did not contain any low M, components that appreciably affected XET activity. To compensate for the low tissue pH of kiwifruit, buffer with a higher pH than that required in the final supematant was used in the initial extraction. To achieve a final homogenate pH of 5.8, Mes (Na+) buffer at pH 6.6 was used for extraction of the OP and IP, buffer at pH 6.2 was used for the core tissues, and buffer at pH 5.8 was used for pea stems (which had little effect on the pH of the extraction buffer). Minor adjustments of the homogenate pH were done with 1 M NaOH or HCl. XET was assayed by its ability to transfer part of a large, nonradioactive xyloglucan (donor) to a reduced [3H]heptasaccharide (acceptor). The method was simple, rapid, and quantitative, and was specific for the transferase function of XET. The standard XET assay was as described by Fry et al. (1992b). The substrate solution contained 0.26% xyloglucan, 0.05 M Mes (Naf), pH 5.8, and 288 kBq/mL [3H]XXXGol.(For pH activity assays, the buffer was omitted.) Reaction mixtures consisted of 20 pL of substrate solution and 20 pL of enzyme extract. Time-course assays were done at 25OC over 1 h with reactions stopped by the addition of 50 p L of 40% formic acid. Each reaction mixture was spotted onto a 3 X 3 cm square of Whatman 3MM paper, which was dried and then washed for 1 h in running tap water to remove unreacted [3H]XXXGol. The paper was dried and polymeric material was assayed for 3H by scintillation counting. Each assay was corrected for the value at time O. XET activities are reported as Bq of 3H-polymer formed per kBq of [3H]XXXGolsupplied. lsolation of C W M and PAW-Soluble Fractions from Kiwifruit
CWM was prepared from 3 g of fresh tissue as described by Redgwell et al. (1992). Briefly, the cryo-milled tissue was homogenized in 8 mL of PAW. The PAW-soluble fraction was dialyzed and the polymers were recovered following freeze drying. The PAW-insoluble residue was extracted in 20 mL of 90% DMSO ovemight to remove starch and the DMSO-insoluble residue (CWM) was washed thoroughly on a glass fiber filter with water and freeze dried.
Xyloglucan and Oligosaccharide Preparation
Xyloglucan was isolated from nasturtium seeds by the method of Rao (1959). The XXXG was prepared from rose cell cultures as described by Lorences and Fry (1993) and converted to its radioactive alditol by reduction with NaB3H4. The solution of [3H]XXXGol (Fry et al., 1993) used for the XET assays had a specific activity of 22.5 MBq/pmol.
Purification of Kiwifruit Xyloglucan and Galactoglucomannan
CWM (3 g) prepared from the harvest fruit was depectinated by sequential extraction in 0.05 M cyclohexane-trans-1, 2-diamine tetra acetate, pH 6.5, and 0.05 M Na2C03.The residue was stirred for 3 h in 8 M KOH containing 20 mM
Xyloglucan Endotransglycosylase in Ripening Kiwifruit
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Table 1. Changes in fruit firmness, SSC, and yield of PAW-soluble polymeric fractions isolated from three tissue zones of kiwifruit at different stages of rbenina Ripening Stage
Harvest l d 2d 4d 5d 6d 15 d (no ethylene treatment)
Fruit Firmness
ssc
kgf
70
9.1 7.2 3.3 2.0 1.3 0.87 7.4
6.9 7.4 9.5 11.5 13.0 14.1 10.8
NaBH4. The KOH-soluble fraction was neutralized and the hemicellulose-rich polysaccharides were recovered after dialysis and freeze drying. The polymers were dissolved in 0.05 M NH4HC03and passed through a column (10 X 2.5 cm) of QAE-50-120 Sephadex (HC03- form) to remove acidic polysaccharides. The 0 . 0 5 - ~NH4HC03 eluate was dialyzed and subjected to barium hydroxide precipitation (Meier, 1965) to separate the galactoglucomannan from the xyloglucan. Traces of xyloglucan were still present in the galactoglucomannan after this procedure and were separated by gel-permeation chromatography on Sephacryl S-400 (1.8 X 120 cm) in pyridine:acetic acid:water (1:1:23, v/v). The absence of Man from the xyloglucan and of Fuc from the galactoglucomannan was taken as evidence of the respective purity of each hemicellulose.
XET Action against Native Substrates
Xyloglucan and galactoglucomannan, purified from the OP of kiwifruit, were tested as donor substrates for kiwifruit XET. The assay was done as described previously, with kiwifruit xyloglucan or galactoglucomannan being used as the nonradioactive donor substrate in place of the nasturtium xyloglucan. A small amount of xyloglucan was present in the kiwifruit XET extract. This acted as a donor substrate and gave a false positive for nonxyloglucan donors being tested. Therefore, before use, the freshly prepared XET extract was passed through a column (0.5 X 2.0 cm) of Sigma microgranular cellulose, which removed the unwanted xyloglucan without diminishing the activity of the XET extract (data not given). CWMs from unripe (harvest) and ripe (6 d after ethylene treatment) fruit were also tested as donor substrates. During ripening significant amounts of CWM (probably mainly pectin)became PAW soluble (Table I), which meant that CWM became enriched in xyloglucan compared with that from unripe fruit. To ensure that at least a comparable amount of xyloglucan was present in the XET assay of CWM from the unripe and ripe CWM, 50% more CWM was taken for the unripe CWM assay. CWM (6 mg unripe, 4 mg ripe) was washed with several changes of Mes (Na+) buffer (pH 5.8) to remove traces of soluble polysaccharide. XET extract (1 mL, postcellulose column) plus the [3H]XXXGol (225 kBq) was added to the buffer-wetted CWMs and the mixtures
PAW-Soluble OP
Core
IP
mg/g fresh weight
1.4 1.2 2.6 2.9 3.4 3.9
2.3 2.4 3.5 3.3 3.6 4.1
1 .a 2.0 2.7 3.4 4.4 5.4
were incubated for 18 h at 25OC. The samples were centrifuged (ZOOOg), the supernatants were discarded, and the CWM residues were washed several times to remove the unincorporated [3H]XXXGol.The amount of 3H in the CWMs was determined by scintillation counting. RESULTS
Following a postharvest ethylene treatment, kiwifruit softened rapidly from 9.1 kgf at harvest to less than 1 kgf 6 d after ethylene treatment (Table I). Tissue from the OP, IP, and core tissues was cryo-milled and separated into CWMand PAW-soluble polymeric fractions. PAW-soluble polymeric fractions contained, in addition to protein, the pectic polysaccharides solubilized in vivo as a result of ripeningrelated alteration of the cell wall (Redgwell et al., 1992). The increase in the weight of the PAW-soluble fraction (Table I), therefore, is an indication of the amount of pectin solubilization occumng as the fruit softened and was consistent with previous results obtained from ethylene-treated fruit. The fresh weights of the OP, IP, and core tissues were 44, 48, and 896, respectively, of the total fresh weight of the fruit. The dry weight of each tissue was 14, 16, and 20% of the fresh weight for the OP, Ir, and core, respectively. Fresh and dry weight ratios for each tissue zone changed little over the 6 d of the experiment. XET in the OP
The induction of ripening in kiwifruit by exogenous ethylene was accompanied by a rapid and pronounced increase in extractable XET activity in the OP. A time-course assay demonstrated linear kinetics over 1 h (Fig. 1). At harvest, XET was just detectable, but 1 d after ethylene treatment there was a 60% increase in activity. As the fruit continued to soften, enzyme activity increased, until 6 d after ethylene treatment, when there was a 12-fold rise compared with levels at harvest. Co-extraction of pea stem tissue with OP from harvest and 6-d fruit tissue showed no inhibition of pea stem XET activity by components of the kiwifruit extact (Table 11). XET Activity in IP and Core
At harvest, extractable XET activities in the IP and core tissues were 4.5 and 42 times higher, respectively, than in
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